Compounds useful as inhibitors of ATR kinase

ABSTRACT

The present invention relates to compounds useful as inhibitors of ATR protein kinase. The invention also relates to pharmaceutically acceptable compositions comprising the compounds of this invention; methods of treating of various diseases, disorders, and conditions using the compounds of this invention; processes for preparing the compounds of this invention; intermediates for the preparation of the compounds of this invention; and methods of using the compounds in in vitro applications, such as the study of kinases in biological and pathological phenomena; the study of intracellular signal transduction pathways mediated by such kinases; and the comparative evaluation of new kinase inhibitors. 
     The compounds of this invention have formula I: 
                         
or a pharmaceutically acceptable salt, wherein the variables are as defined herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit under 35 U.S.C. §119 UnitedStates of Provisional Application No. 61/714,238, filed Oct. 16, 2012,as well as Provisional Application No. 61/757,792, filed Jan. 29, 2013.

SEQUENCE LISTING

The instant application contains a Sequence Listing in .txt format whichhas been submitted via EFS-Web and is hereby incorporated by referencein its entirety. The Sequence Listing, created on Oct. 1, 2014, is namedVPI12-133US.txt and is 677 bytes in size.

BACKGROUND OF THE INVENTION

ATR (“ATM and Rad3 related”) kinase is a protein kinase involved incellular responses to certain forms of DNA damage (eg double strandbreaks and replication stress). ATR kinase acts with ATM (“ataxiatelangiectasia mutated”) kinase and many other proteins to regulate acell's response to double strand DNA breaks and replication stress,commonly referred to as the DNA Damage Response (“DDR”). The DDRstimulates DNA repair, promotes survival and stalls cell cycleprogression by activating cell cycle checkpoints, which provide time forrepair. Without the DDR, cells are much more sensitive to DNA damage andreadily die from DNA lesions induced by endogenous cellular processessuch as DNA replication or exogenous DNA damaging agents commonly usedin cancer therapy.

Healthy cells can rely on a host of different proteins for DNA repairincluding the DDR kinases ATR and ATM. In some cases these proteins cancompensate for one another by activating functionally redundant DNArepair processes. On the contrary, many cancer cells harbour defects insome of their DNA repair processes, such as ATM signaling, and thereforedisplay a greater reliance on their remaining intact DNA repair proteinswhich include ATR.

In addition, many cancer cells express activated oncogenes or lack keytumour suppressors, and this can make these cancer cells prone todysregulated phases of DNA replication which in turn cause DNA damage.ATR has been implicated as a critical component of the DDR in responseto disrupted DNA replication. As a result, these cancer cells are moredependent on ATR activity for survival than healthy cells. Accordingly,ATR inhibitors may be useful for cancer treatment, either used alone orin combination with DNA damaging agents, because they shut down a DNArepair mechanism that is more important for cellular survival in manycancer cells than in healthy normal cells.

In fact, disruption of ATR function (e.g. by gene deletion) has beenshown to promote cancer cell death both in the absence and presence ofDNA damaging agents. This suggests that ATR inhibitors may be effectiveboth as single agents and as potent sensitizers to radiotherapy orgenotoxic chemotherapy.

ATR peptide can be expressed and isolated using a variety of methodsknown in the literature (see e.g., Ünsal-Kaçmaz et al, PNAS 99: 10, pp6673-6678, May 14, 2002; see also Kumagai et al. Cell 124, pp 943-955,Mar. 10, 2006; Unsal-Kacmaz et al. Molecular and Cellular Biology,February 2004, p 1292-1300; and Hall-Jackson et al. Oncogene 1999, 18,6707-6713).

For all of these reasons, there is a need for the development of potentand selective ATR inhibitors for the treatment of cancer, either assingle agents or as combination therapies with radiotherapy or genotoxicchemotherapy.

SUMMARY OF THE INVENTION

The present invention relates to compounds useful as inhibitors of ATRprotein kinase. The invention also relates to pharmaceuticallyacceptable compositions comprising the compounds of this invention;methods of treating of various diseases, disorders, and conditions usingthe compounds of this invention; processes for preparing the compoundsof this invention; intermediates for the preparation of the compounds ofthis invention; and methods of using the compounds in in vitroapplications, such as the study of kinases in biological andpathological phenomena; the study of intracellular signal transductionpathways mediated by such kinases; and the comparative evaluation of newkinase inhibitors.

The compounds of the invention are very potent ATR inhibitors. Thesecompounds have an unexpected ability to treat cancer as single agents.These compounds also show surprising synergy with other cancer agents,such as cisplatin and gemcitabine, in combination therapies.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention provides a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein:

-   R¹ is independently selected from a C₁₋₆ aliphatic, a C₃₋₆    cycloaliphatic, or a 3-6 membered heterocyclyl having 1-2    heteroatoms selected from O, N, or S;-   R² is independently selected from H, C₁₋₃aliphatic or a    C₃₋₆cycloaliphatic;-   R³ is independently selected from H, C₁₋₆aliphatic,    C₃₋₆cycloaliphatic, or a 3-6 membered heterocyclyl having 1-2    heteroatoms selected from O, N, or S;-   R⁴ is independently selected from H, C₁₋₆aliphatic,    C₃₋₆cycloaliphatic, or a 3-6 membered heterocyclyl having 1-2    heteroatoms selected from O, N, or S; or-   R² and R³, together with the atoms to which they are attached, form    an optionally substituted 4-7 membered heterocyclic ring having 1-2    heteroatoms independently selected from O, N, or S; or-   R³ and R⁴, together with the nitrogen atom to which they are    attached, form an optionally substituted 4-7 membered heterocyclic    ring having 1-2 heteroatoms selected from O, N, or S.

In some embodiments, R¹ is a C₁₋₆aliphatic. In another embodiment, R¹ isan isopropyl.

In another embodiment, R² is H.

In yet another embodiment, R⁴ is H or C₁₋₆aliphatic. In someembodiments, R⁴ is H. In other embodiments, R⁴ is methyl.

In another embodiment, R³ is C₁₋₆aliphatic. In yet another embodiment,R³ is methyl.

In some embodiments, R² and R³, together with the atoms to which theyare attached, form an optionally substituted 4-7 membered heterocyclicring having 1-2 heteroatoms independently selected from O, N, or S. Inother embodiments, R² and R³, together with the atoms to which they areattached, form an optionally substituted 4-7 membered heterocyclic ringselected from pyrrolidinyl, azetidinyl, or piperidinyl. In anotherembodiment, the 4-7 membered heterocyclic ring formed by R² and R³ isoptionally substituted with halo, NH₂, CN, C₁₋₆aliphatic, [C₆ aryl orphenyl], 5-6 membered heteroaryl, C₄₋₇carbocyclyl, or 4-7 memberedheterocyclyl.

In another embodiment R³ and R⁴, together with the nitrogen atom towhich they are attached, form an optionally substituted 4-7 memberedheterocyclic ring having 1-2 heteroatoms independently selected from O,N, or S. In another embodiment, the 4-7 membered heterocyclic ringformed by R³ and R⁴ is optionally substituted with halo, CN, NH₂,C₁₋₆aliphatic, C₅₋₆aryl, 5-6 membered heteroaryl, C₄₋₇carbocyclyl, or4-7 membered heterocyclyl.

In some embodiments, the variables are as depicted in the compounds ofthe disclosure including compounds in table 1 below.

TABLE 1 Number Structure I-1

I-2

I-3

I-4

I-6

I-7

Compounds of this invention include those described generally herein,and are further illustrated by the classes, subclasses, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated. For purposes of this invention, the chemicalelements are identified in accordance with the Periodic Table of theElements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed.Additionally, general principles of organic chemistry are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5^(th) Ed.,Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference.

As described herein, a specified number range of atoms includes anyinteger therein. For example, a group having from 1-4 atoms could have1, 2, 3, or 4 atoms.

As described herein, compounds of the invention may optionally besubstituted with one or more substituents, such as are illustratedgenerally herein, or as exemplified by particular classes, subclasses,and species of the invention. It will be appreciated that the phrase“optionally substituted” is used interchangeably with the phrase“substituted or unsubstituted.” In general, the term “substituted”,whether preceded by the term “optionally” or not, refers to thereplacement of hydrogen radicals in a given structure with the radicalof a specified substituent. Unless otherwise indicated, an optionallysubstituted group may have a substituent at each substitutable positionof the group, and when more than one position in any given structure maybe substituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds.

Unless otherwise indicated, a substituent connected by a bond drawn fromthe center of a ring means that the substituent can be bonded to anyposition in the ring. In example i below, for instance, J¹ can be bondedto any position on the pyridyl ring. For bicyclic rings, a bond drawnthrough both rings indicates that the substituent can be bonded from anyposition of the bicyclic ring. In example ii below, for instance, J¹ canbe bonded to the 5-membered ring (on the nitrogen atom, for instance),and to the 6-membered ring.

The term “stable”, as used herein, refers to compounds that are notsubstantially altered when subjected to conditions to allow for theirproduction, detection, recovery, purification, and use for one or moreof the purposes disclosed herein. In some embodiments, a stable compoundor chemically feasible compound is one that is not substantially alteredwhen kept at a temperature of 40° C. or less, in the absence of moistureor other chemically reactive conditions, for at least a week.

The term “aliphatic” or “aliphatic group”, as used herein, means astraight-chain (i.e., unbranched), branched, or cyclic, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation that has a single point ofattachment to the rest of the molecule.

Unless otherwise specified, aliphatic groups contain 1-20 aliphaticcarbon atoms. In some embodiments, aliphatic groups contain 1-10aliphatic carbon atoms. In other embodiments, aliphatic groups contain1-8 aliphatic carbon atoms. In still other embodiments, aliphatic groupscontain 1-6 aliphatic carbon atoms, and in yet other embodimentsaliphatic groups contain 1-4 aliphatic carbon atoms. Aliphatic groupsmay be linear or branched, substituted or unsubstituted alkyl, alkenyl,or alkynyl groups. Specific examples include, but are not limited to,methyl, ethyl, isopropyl, n-propyl, sec-butyl, vinyl, n-butenyl,ethynyl, and tert-butyl. Aliphatic groups may also be cyclic, or have acombination of linear or branched and cyclic groups. Examples of suchtypes of aliphatic groups include, but are not limited to cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, —CH₂-cyclopropyl,CH₂CH₂CH(CH₃)-cyclohexyl.

The term “cycloaliphatic” (or “carbocycle” or “carbocyclyl”) refers to amonocyclic C₃-C₈ hydrocarbon or bicyclic C₈-C₁₂ hydrocarbon that iscompletely saturated or that contains one or more units of unsaturation,but which is not aromatic, that has a single point of attachment to therest of the molecule wherein any individual ring in said bicyclic ringsystem has 3-7 members. Examples of cycloaliphatic groups include, butare not limited to, cycloalkyl and cycloalkenyl groups. Specificexamples include, but are not limited to, cyclohexyl, cyclopropenyl, andcyclobutyl.

The term “heterocycle”, “heterocyclyl”, or “heterocyclic” as used hereinmeans non-aromatic, monocyclic, bicyclic, or tricyclic ring systems inwhich one or more ring members are an independently selected heteroatom.In some embodiments, the “heterocycle”, “heterocyclyl”, or“heterocyclic” group has three to fourteen ring members in which one ormore ring members is a heteroatom independently selected from oxygen,sulfur, nitrogen, or phosphorus, and each ring in the system contains 3to 7 ring members.

Examples of heterocycles include, but are not limited to,3-1H-benzimidazol-2-one, 3-(1-alkyl)-benzimidazol-2-one,2-tetrahydrofuranyl, 3-tetrahydrofuranyl, 2-tetrahydrothiophenyl,3-tetrahydrothiophenyl, 2-morpholino, 3-morpholino, 4-morpholino,2-thiomorpholino, 3-thiomorpholino, 4-thiomorpholino, 1-pyrrolidinyl,2-pyrrolidinyl, 3-pyrrolidinyl, 1-tetrahydropiperazinyl,2-tetrahydropiperazinyl, 3-tetrahydropiperazinyl, 1-piperidinyl,2-piperidinyl, 3-piperidinyl, 1-pyrazolinyl, 3-pyrazolinyl,4-pyrazolinyl, 5-pyrazolinyl, 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-piperidinyl, 2-thiazolidinyl, 3-thiazolidinyl,4-thiazolidinyl, 1-imidazolidinyl, 2-imidazolidinyl, 4-imidazolidinyl,5-imidazolidinyl, indolinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, benzothiolane, benzodithiane, and1,3-dihydro-imidazol-2-one.

Cyclic groups, (e.g. cycloaliphatic and heterocycles), can be linearlyfused, bridged, or spirocyclic.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen,phosphorus, or silicon (including, any oxidized form of nitrogen,sulfur, phosphorus, or silicon; the quaternized form of any basicnitrogen or; a substitutable nitrogen of a heterocyclic ring, forexample N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) orNR⁺ (as in N-substituted pyrrolidinyl)).

The term “unsaturated”, as used herein, means that a moiety has one ormore units of unsaturation. As would be known by one of skill in theart, unsaturated groups can be partially unsaturated or fullyunsaturated. Examples of partially unsaturated groups include, but arenot limited to, butene, cyclohexene, and tetrahydropyridine. Fullyunsaturated groups can be aromatic, anti-aromatic, or non-aromatic.Examples of fully unsaturated groups include, but are not limited to,phenyl, cyclooctatetraene, pyridyl, thienyl, and1-methylpyridin-2(1H)-one.

The term “alkoxy”, or “thioalkyl”, as used herein, refers to an alkylgroup, as previously defined, attached through an oxygen (“alkoxy”) orsulfur (“thioalkyl”) atom.

The terms “haloalkyl”, “haloalkenyl”, “haloaliphatic”, and “haloalkoxy”mean alkyl, alkenyl or alkoxy, as the case may be, substituted with oneor more halogen atoms. This term includes perfluorinated alkyl groups,such as —CF₃ and —CF₂CF₃.

The terms “halogen”, “halo”, and “hal” mean F, Cl, Br, or I.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic,bicyclic, and tricyclic ring systems having a total of five to fourteenring members, wherein at least one ring in the system is aromatic andwherein each ring in the system contains 3 to 7 ring members. The term“aryl” may be used interchangeably with the term “aryl ring”.

The term “heteroaryl”, used alone or as part of a larger moiety as in“heteroaralkyl” or “heteroarylalkoxy”, refers to monocyclic, bicyclic,and tricyclic ring systems having a total of five to fourteen ringmembers, wherein at least one ring in the system is aromatic, at leastone ring in the system contains one or more heteroatoms, and whereineach ring in the system contains 3 to 7 ring members. The term“heteroaryl” may be used interchangeably with the term “heteroaryl ring”or the term “heteroaromatic”. Examples of heteroaryl rings include, butare not limited to, 2-furanyl, 3-furanyl, N-imidazolyl, 2-imidazolyl,4-imidazolyl, 5-imidazolyl, benzimidazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, N-pyrrolyl,2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl,4-pyrimidinyl, 5-pyrimidinyl, pyridazinyl (e.g., 3-pyridazinyl),2-thiazolyl, 4-thiazolyl, 5-thiazolyl, tetrazolyl (e.g., 5-tetrazolyl),triazolyl (e.g., 2-triazolyl and 5-triazolyl), 2-thienyl, 3-thienyl,benzofuryl, benzothiophenyl, indolyl (e.g., 2-indolyl), pyrazolyl (e.g.,2-pyrazolyl), isothiazolyl, 1,2,3-oxadiazolyl, 1,2,5-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,3-triazolyl, 1,2,3-thiadiazolyl,1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, purinyl, pyrazinyl,1,3,5-triazinyl, quinolinyl (e.g., 2-quinolinyl, 3-quinolinyl,4-quinolinyl), and isoquinolinyl (e.g., 1-isoquinolinyl,3-isoquinolinyl, or 4-isoquinolinyl).

It shall be understood that the term “heteroaryl” includes certain typesof heteroaryl rings that exist in equilibrium between two differentforms. More specifically, for example, species such hydropyridine andpyridinone (and likewise hydroxypyrimidine and pyrimidinone) are meantto be encompassed within the definition of “heteroaryl.”

The term “protecting group” and “protective group” as used herein, areinterchangeable and refer to an agent used to temporarily block one ormore desired functional groups in a compound with multiple reactivesites. In certain embodiments, a protecting group has one or more, orpreferably all, of the following characteristics: a) is addedselectively to a functional group in good yield to give a protectedsubstrate that is b) stable to reactions occurring at one or more of theother reactive sites; and c) is selectively removable in good yield byreagents that do not attack the regenerated, deprotected functionalgroup. As would be understood by one skilled in the art, in some cases,the reagents do not attack other reactive groups in the compound. Inother cases, the reagents may also react with other reactive groups inthe compound. Examples of protecting groups are detailed in Greene, T.W., Wuts, P. G in “Protective Groups in Organic Synthesis”, ThirdEdition, John Wiley & Sons, New York: 1999 (and other editions of thebook), the entire contents of which are hereby incorporated byreference. The term “nitrogen protecting group”, as used herein, refersto an agent used to temporarily block one or more desired nitrogenreactive sites in a multifunctional compound. Preferred nitrogenprotecting groups also possess the characteristics exemplified for aprotecting group above, and certain exemplary nitrogen protecting groupsare also detailed in Chapter 7 in Greene, T. W., Wuts, P. G in“Protective Groups in Organic Synthesis”, Third Edition, John Wiley &Sons, New York: 1999, the entire contents of which are herebyincorporated by reference.

In some embodiments, a methylene unit of an alkyl or aliphatic chain isoptionally replaced with another atom or group. Examples of such atomsor groups include, but are not limited to, nitrogen, oxygen, sulfur,—C(O)—, —C(═N—CN)—, —C(═NR)—, —C(═NOR)—, —SO—, and —SO₂—. These atoms orgroups can be combined to form larger groups. Examples of such largergroups include, but are not limited to, —OC(O)—, —C(O)CO—, —CO₂—,—C(O)NR—, —C(═N—CN), —NRCO—, —NRC(O)O—, —SO₂NR—, —NRSO₂—, —NRC(O)NR—,—OC(O)NR—, and —NRSO₂NR—, wherein R is, for example, H or C₁₋₆aliphatic.It should be understood that these groups can be bonded to the methyleneunits of the aliphatic chain via single, double, or triple bonds. Anexample of an optional replacement (nitrogen atom in this case) that isbonded to the aliphatic chain via a double bond would be —CH₂CH═N—CH₃.In some cases, especially on the terminal end, an optional replacementcan be bonded to the aliphatic group via a triple bond. One example ofthis would be CH₂CH₂CH₂C≡N. It should be understood that in thissituation, the terminal nitrogen is not bonded to another atom.

It should also be understood that, the term “methylene unit” can alsorefer to branched or substituted methylene units. For example, in anisopropyl moiety [—CH(CH₃)₂], a nitrogen atom (e.g. NR) replacing thefirst recited “methylene unit” would result in dimethylamine [—N(CH₃)₂].In instances such as these, one of skill in the art would understandthat the nitrogen atom will not have any additional atoms bonded to it,and the “R” from “NR” would be absent in this case.

Unless otherwise indicated, the optional replacements form a chemicallystable compound. Optional replacements can occur both within the chainand/or at either end of the chain; i.e. both at the point of attachmentand/or also at the terminal end. Two optional replacements can also beadjacent to each other within a chain so long as it results in achemically stable compound. For example, a C₃ aliphatic can beoptionally replaced by 2 nitrogen atoms to form —C—N≡N. The optionalreplacements can also completely replace all of the carbon atoms in achain. For example, a C₃ aliphatic can be optionally replaced by —NR—,—C(O)—, and —NR— to form —NRC(O)NR— (a urea).

Unless otherwise indicated, if the replacement occurs at the terminalend, the replacement atom is bound to a hydrogen atom on the terminalend. For example, if a methylene unit of —CH₂CH₂CH₃ were optionallyreplaced with —O—, the resulting compound could be —OCH₂CH₃, —CH₂OCH₃,or —CH₂CH₂OH. It should be understood that if the terminal atom does notcontain any free valence electrons, then a hydrogen atom is not requiredat the terminal end (e.g., —CH₂CH₂CH═O or —CH₂CH₂C≡N).

Unless otherwise indicated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, geometric,conformational, and rotational) forms of the structure. For example, theR and S configurations for each asymmetric center, (Z) and (E) doublebond isomers, and (Z) and (E) conformational isomers are included inthis invention. As would be understood to one skilled in the art, asubstituent can freely rotate around any rotatable bonds. For example, asubstituent drawn as

also represents

Therefore, single stereochemical isomers as well as enantiomeric,diastereomeric, geometric, conformational, and rotational mixtures ofthe present compounds are within the scope of the invention.

Unless otherwise indicated, all tautomeric forms of the compounds of theinvention are within the scope of the invention.

Additionally, unless otherwise indicated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures except for the replacement of hydrogen by deuteriumor tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enrichedcarbon are within the scope of this invention. Such compounds areuseful, for example, as analytical tools or probes in biological assays.

Pharmaceutically Acceptable Salts

The compounds of this invention can exist in free form for treatment, orwhere appropriate, as a pharmaceutically acceptable salt.

A “pharmaceutically acceptable salt” means any non-toxic salt of acompound of this invention that, upon administration to a recipient, iscapable of providing, either directly or indirectly, a compound of thisinvention or an inhibitorily active metabolite or residue thereof Asused herein, the term “inhibitorily active metabolite or residuethereof” means that a metabolite or residue thereof is also an inhibitorof the ATR protein kinase.

Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge et al., describe pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporatedherein by reference. Pharmaceutically acceptable salts of the compoundsof this invention include those derived from suitable inorganic andorganic acids and bases. These salts can be prepared in situ during thefinal isolation and purification of the compounds. Acid addition saltscan be prepared by 1) reacting the purified compound in its free-basedform with a suitable organic or inorganic acid and 2) isolating the saltthus formed.

Examples of pharmaceutically acceptable, nontoxic acid addition saltsare salts of an amino group formed with inorganic acids such ashydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid andperchloric acid or with organic acids such as acetic acid, oxalic acid,maleic acid, tartaric acid, citric acid, succinic acid or malonic acidor by using other methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, glycolate, gluconate, glycolate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate,lauryl sulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, salicylate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like.

Base addition salts can be prepared by 1) reacting the purified compoundin its acid form with a suitable organic or inorganic base and 2)isolating the salt thus formed. Salts derived from appropriate basesinclude alkali metal (e.g., sodium, lithium, and potassium), alkalineearth metal (e.g., magnesium and calcium), ammonium and N⁺(C₁₋₄alkyl)₄salts. This invention also envisions the quaternization of any basicnitrogen-containing groups of the compounds disclosed herein. Water oroil-soluble or dispersible products may be obtained by suchquaternization.

Further pharmaceutically acceptable salts include, when appropriate,nontoxic ammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate and aryl sulfonate. Other acids and bases,while not in themselves pharmaceutically acceptable, may be employed inthe preparation of salts useful as intermediates in obtaining thecompounds of the invention and their pharmaceutically acceptable acid orbase addition salts.

Abbreviations

The following abbreviations are used:

-   DMSO dimethyl sulfoxide-   ATP adenosine triphosphate-   ¹HNMR proton nuclear magnetic resonance-   HPLC high performance liquid chromatography-   LCMS liquid chromatography-mass spectrometry-   TLC thin layer chromatography-   Rt retention time    Compound Uses

One aspect of this invention provides compounds that are inhibitors ofATR kinase, and thus are useful for treating or lessening the severityof a disease, condition, or disorder where ATR is implicated in thedisease, condition, or disorder.

Another aspect of this invention provides compounds that are useful forthe treatment of diseases, disorders, and conditions characterized byexcessive or abnormal cell proliferation. Such diseases include, aproliferative or hyperproliferative disease. Examples of proliferativeand hyperproliferative diseases include, without limitation, cancer andmyeloproliferative disorders.

In some embodiments, said compounds are selected from the groupconsisting of a compound of formula I. The term “cancer” includes, butis not limited to the following cancers. Oral: buccal cavity, lip,tongue, mouth, pharynx; Cardiac: sarcoma (angiosarcoma, fibrosarcoma,rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma andteratoma; Lung: non-small cell, bronchogenic carcinoma (squamous cell orepidermoid, undifferentiated small cell, undifferentiated large cell,adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma,sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;Gastrointestinal: esophagus (squamous cell carcinoma, larynx,adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel or smallintestines (adenocarcinoma, lymphoma, carcinoid tumors, Karposi'ssarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), largebowel or large intestines (adenocarcinoma, tubular adenoma, villousadenoma, hamartoma, leiomyoma), colon, colon-rectum, colorectal; rectum,Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor[nephroblastoma], lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma, biliary passages;Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myeloma, malignant giant cell tumorchordoma, osteochronfroma (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors; Nervous system: skull (osteoma, hemangioma,granuloma, xanthoma, osteitis deformans), meninges (meningioma,meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma,glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform,oligodendroglioma, schwannoma, retinoblastoma, congenital tumors),spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological:uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumorcervical dysplasia), ovaries (ovarian carcinoma [serouscystadenocarcinoma, mucinous cystadenocarcinoma, unclassifiedcarcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma(embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast;Hematologic: blood (myeloid leukemia [acute and chronic], acutelymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferativediseases, multiple myeloma, myelodysplastic syndrome), Hodgkin'sdisease, non-Hodgkin's lymphoma [malignant lymphoma] hairy cell;lymphoid disorders; Skin: malignant melanoma, basal cell carcinoma,squamous cell carcinoma, Karposi's sarcoma, keratoacanthoma, molesdysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis,Thyroid gland: papillary thyroid carcinoma, follicular thyroidcarcinoma, undifferentiated thyroid cancer, medullary thyroid carcinoma,multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type2B, familial medullary thyroid cancer, pheochromocytoma, paraganglioma;and Adrenal glands: neuroblastoma.

In some embodiments, the cancer is lung cancer. In other embodiments,the lung cancer is non-small cell lung cancer or small cell lung cancer.In another embodiment, the cancer is non-small cell lung cancer. In yetanother embodiment, the non-small cell lung cancer is squamous non-smallcell lung cancer.

In some embodiments, the cancer is selected from a cancer of the lung orthe pancreas. In other embodiments, the cancer is selected from lungcancer, head and neck cancer, pancreatic cancer, gastric cancer, orbrain cancer. In yet other embodiments, the cancer is selected fromnon-small cell lung cancer, small cell lung cancer, pancreatic cancer,biliary tract cancer, head and neck cancer, bladder cancer, colorectalcancer, glioblastoma, esophageal cancer, breast cancer, hepatocellularcarcinoma, or ovarian cancer.

In some embodiments, the cancer is selected from colorectal, thyroid, orbreast cancer. In other embodiments, the cancer is triple negativebreast cancer.

Thus, the term “cancerous cell” as provided herein, includes a cellafflicted by any one of the above-identified conditions. In someembodiments, the cancer is selected from colorectal, thyroid, or breastcancer.

The term “myeloproliferative disorders”, includes disorders such aspolycythemia vera, thrombocythemia, myeloid metaplasia withmyelofibrosis, hypereosinophilic syndrome, juvenile myelomonocyticleukemia, systemic mast cell disease, and hematopoietic disorders, inparticular, acute-myelogenous leukemia (AML), chronic-myelogenousleukemia (CML), acute-promyelocytic leukemia (APL), and acutelymphocytic leukemia (ALL).

Pharmaceutically Acceptable Derivatives or Prodrugs

In addition to the compounds of this invention, pharmaceuticallyacceptable derivatives or prodrugs of the compounds of this inventionmay also be employed in compositions to treat or prevent the hereinidentified disorders.

The compounds of this invention can also exist as pharmaceuticallyacceptable derivatives.

A “pharmaceutically acceptable derivative” is an adduct or derivativewhich, upon administration to a patient in need, is capable ofproviding, directly or indirectly, a compound as otherwise describedherein, or a metabolite or residue thereof Examples of pharmaceuticallyacceptable derivatives include, but are not limited to, esters and saltsof such esters.

A “pharmaceutically acceptable derivative or prodrug” means anypharmaceutically acceptable ester, salt of an ester or other derivativeor salt thereof of a compound, of this invention which, uponadministration to a recipient, is capable of providing, either directlyor indirectly, a compound of this invention or an inhibitorily activemetabolite or residue thereof Particularly favoured derivatives orprodrugs are those that increase the bioavailability of the compounds ofthis invention when such compounds are administered to a patient (e.g.,by allowing an orally administered compound to be more readily absorbedinto the blood) or which enhance delivery of the parent compound to abiological compartment (e.g., the brain or lymphatic system) relative tothe parent species.

Pharmaceutically acceptable prodrugs of the compounds of this inventioninclude, without limitation, esters, amino acid esters, phosphateesters, metal salts and sulfonate esters.

Pharmaceutical Compositions

The present invention also provides compounds and compositions that areuseful as inhibitors of ATR kinase.

One aspect of this invention provides pharmaceutically acceptablecompositions that comprise any of the compounds as described herein, andoptionally comprise a pharmaceutically acceptable carrier, adjuvant orvehicle.

The pharmaceutically acceptable carrier, adjuvant, or vehicle, as usedherein, includes any and all solvents, diluents, or other liquidvehicle, dispersion or suspension aids, surface active agents, isotonicagents, thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W.Martin (Mack Publishing Co., Easton, Pa., 1980) discloses variouscarriers used in formulating pharmaceutically acceptable compositionsand known techniques for the preparation thereof Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutically acceptable composition, its use iscontemplated to be within the scope of this invention.

Some examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, or potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, wool fat, sugars such aslactose, glucose and sucrose; starches such as corn starch and potatostarch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; powdered tragacanth;malt; gelatin; talc; excipients such as cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil; safflower oil; sesameoil; olive oil; corn oil and soybean oil; glycols; such a propyleneglycol or polyethylene glycol; esters such as ethyl oleate and ethyllaurate; agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

Combination Therapies

Another aspect of this invention is directed towards a method oftreating cancer in a subject in need thereof, comprising administrationof a compound of this invention or a pharmaceutically acceptable saltthereof, and an additional therapeutic agent. In some embodiments, saidmethod comprises the sequential or co-administration of the compound ora pharmaceutically acceptable salt thereof, and the additionaltherapeutic agent.

In some embodiments, said additional therapeutic agent is an anti-canceragent. In other embodiments, said additional therapeutic agent is aDNA-damaging agent. In yet other embodiments, said additionaltherapeutic agent is selected from radiation therapy, chemotherapy, orother agents typically used in combination with radiation therapy orchemotherapy, such as radiosensitizers and chemosensitizers. In yetother embodiments, said additional therapeutic agent is ionizingradiation.

As would be known by one of skill in the art, radiosensitizers areagents that can be used in combination with radiation therapy.Radiosensitizers work in various different ways, including, but notlimited to, making cancer cells more sensitive to radiation therapy,working in synergy with radiation therapy to provide an improvedsynergistic effect, acting additively with radiation therapy, orprotecting surrounding healthy cells from damage caused by radiationtherapy. Likewise chemosensitizers are agents that can be used incombination with chemotherapy. Similarly, chemosensitizers work invarious different ways, including, but not limited to, making cancercells more sensitive to chemotherapy, working in synergy withchemotherapy to provide an improved synergistic effect, actingadditively to chemotherapy, or protecting surrounding healthy cells fromdamage caused by chemotherapy.

Examples of DNA-damaging agents that may be used in combination withcompounds of this invention include, but are not limited to Platinatingagents, such as Carboplatin, Nedaplatin, Satraplatin and otherderivatives; Topo I inhibitors, such as Topotecan, irinotecan/SN38,rubitecan and other derivatives; Antimetabolites, such as Folic family(Methotrexate, Pemetrexed and relatives); Purine antagonists andPyrimidine antagonists (Thioguanine, Fludarabine, Cladribine,Cytarabine, Gemcitabine, 6-Mercaptopurine, 5-Fluorouracil (5FU) andrelatives); Alkylating agents, such as Nitrogen mustards(Cyclophosphamide, Melphalan, Chlorambucil, mechlorethamine, Ifosfamideand relatives); nitrosoureas (eg Carmustine); Triazenes (Dacarbazine,temozolomide); Alkyl sulphonates (eg Busulfan); Procarbazine andAziridines; Antibiotics, such as Hydroxyurea, Anthracyclines(doxorubicin, daunorubicin, epirubicin and other derivatives);Anthracenediones (Mitoxantrone and relatives); Streptomyces family(Bleomycin, Mitomycin C, actinomycin); and Ultraviolet light.

Other therapies or anticancer agents that may be used in combinationwith the inventive agents of the present invention include surgery,radiotherapy (in but a few examples, gamma-radiation, neutron beamradiotherapy, electron beam radiotherapy, proton therapy, brachytherapy,and systemic radioactive isotopes, to name a few), endocrine therapy,biologic response modifiers (interferons, interleukins, and tumornecrosis factor (TNF) to name a few), hyperthermia and cryotherapy,agents to attenuate any adverse effects (e.g., antiemetics), and otherapproved chemotherapeutic drugs, including, but not limited to, the DNAdamaging agents listed herein, spindle poisons (Vinblastine,Vincristine, Vinorelbine, Paclitaxel), podophyllotoxins (Etoposide,Irinotecan, Topotecan), nitrosoureas (Carmustine, Lomustine), inorganicions (Cisplatin, Carboplatin), enzymes (Asparaginase), and hormones(Tamoxifen, Leuprolide, Flutamide, and Megestrol), Gleevec™, adriamycin,dexamethasone, and cyclophosphamide.

A compound of the instant invention may also be useful for treatingcancer in combination with any of the following therapeutic agents:abarelix (Plenaxis depot®); aldesleukin (Prokine®); Aldesleukin(Proleukin®); Alemtuzumabb (Campath®); alitretinoin (Panretin®);allopurinol (Zyloprim®); altretamine (Hexalen®); amifostine (Ethyol®);anastrozole (Arimidex®); arsenic trioxide (Trisenox®); asparaginase(Elspar®); azacitidine (Vidaza®); bevacuzimab (Avastin®); bexarotenecapsules (Targretin®); bexarotene gel (Targretin®); bleomycin(Blenoxane®); bortezomib (Velcade®); busulfan intravenous (Busulfex®);busulfan oral (Myleran®); calusterone (Methosarb®); capecitabine(Xeloda®); carboplatin (Paraplatin®); carmustine (BCNU®, BiCNU®);carmustine (Gliadel®); carmustine with Polifeprosan 20 Implant (GliadelWafer®); celecoxib (Celebrex®); cetuximab (Erbitux®); chlorambucil(Leukeran®); cisplatin (Platinol®); cladribine (Leustatin 2-CdA®);clofarabine (Clolar®); cyclophosphamide (Cytoxan®, Neosar®);cyclophosphamide (Cytoxan Injection®); cyclophosphamide (CytoxanTablet®); cytarabine (Cytosar-U®); cytarabine liposomal (DepoCyt®);dacarbazine (DTIC-Dome®); dactinomycin, actinomycin D (Cosmegen®);Darbepoetin alfa (Aranesp®); daunorubicin liposomal (DanuoXome®);daunorubicin, daunomycin (Daunorubicin®); daunorubicin, daunomycin(Cerubidine®); Denileukin diftitox (Ontak®); dexrazoxane (Zinecard®);docetaxel (Taxotere®); doxorubicin (Adriamycin PFS®); doxorubicin(Adriamycin®, Rubex®); doxorubicin (Adriamycin PFS Injection®);doxorubicin liposomal (Doxil®); dromostanolone propionate(dromostanolone®); dromostanolone propionate (masterone injection®);Elliott's B Solution (Elliott's B Solution®); epirubicin (Ellence®);Epoetin alfa (epogen®); erlotinib (Tarceva®); estramustine (Emcyt®);etoposide phosphate (Etopophos®); etoposide, VP-16 (Vepesid®);exemestane (Aromasin®); Filgrastim (Neupogen®); floxuridine(intraarterial) (FUDR®); fludarabine (Fludara®); fluorouracil, 5-FU(Adrucil®); fulvestrant (Faslodex®); gefitinib (Iressa®); gemcitabine(Gemzar®); gemtuzumab ozogamicin (Mylotarg®); goserelin acetate (ZoladexImplant®); goserelin acetate (Zoladex®); histrelin acetate (Histrelinimplant®); hydroxyurea (Hydrea®); Ibritumomab Tiuxetan (Zevalin®);idarubicin (Idamycin®); ifosfamide (IFEX®); imatinib mesylate(Gleevec®); interferon alfa 2a (Roferon A®); Interferon alfa-2b (IntronA®); irinotecan (Camptosar®); lenalidomide (Revlimid®); letrozole(Ferrara®); leucovorin (Wellcovorin®, Leucovorin®); Leuprolide Acetate(Eligard®); levamisole (Ergamisol®); lomustine, CCNU (CeeBU®);meclorethamine, nitrogen mustard (Mustargen®); megestrol acetate(Megace®); melphalan, L-PAM (Alkeran®); mercaptopurine, 6-MP(Purinethol®); mesna (Mesnex®); mesna (Mesnex tabs®); methotrexate(Methotrexate®); methoxsalen (Uvadex®); mitomycin C (Mutamycin®);mitotane (Lysodren®); mitoxantrone (Novantrone®); nandrolonephenpropionate (Durabolin-50®); nelarabine (Arranon®); Nofetumomab(Verluma®); Oprelvekin (Neumega®); oxaliplatin (Eloxatin®); paclitaxel(Paxene®); paclitaxel (Taxol®); paclitaxel protein-bound particles(Abraxane®); palifermin (Kepivance®); pamidronate (Aredia®); pegademase(Adagen (Pegademase Bovine)®); pegaspargase (Oncaspar®); Pegfilgrastim(Neulasta®); pemetrexed disodium (Alimta®); pentostatin (Nipent®);pipobroman (Vercyte®); plicamycin, mithramycin (Mithracin®); porfimersodium (Photofrin®); procarbazine (Matulane®); quinacrine (Atabrine®);Rasburicase (Elitek®); Rituximab (Rituxan®); sargramostim (Leukine®);Sargramostim (Prokine®); sorafenib (Nexavar®); streptozocin (Zanosar®);sunitinib maleate (Sutent®); talc (Sclerosol®); tamoxifen (Nolvadex®);temozolomide (Temodar®); teniposide, VM-26 (Vumon®); testolactone(Teslac®); thioguanine, 6-TG (Thioguanine®); thiotepa (Thioplex®);topotecan (Hycamtin®); toremifene (Fareston®); Tositumomab (Bexxar®);Tositumomab/I-131 tositumomab (Bexxar®); Trastuzumab (Herceptin®);tretinoin, ATRA (Vesanoid®); Uracil Mustard (Uracil Mustard Capsules®);valrubicin (Valstar®); vinblastine (Velban®); vincristine (Oncovin®);vinorelbine (Navelbine®); zoledronate (Zometa®) and vorinostat(Zolinza®).

For a comprehensive discussion of updated cancer therapies see,http://www.nci.nih.gov/, a list of the FDA approved oncology drugs athttp://www.fda.gov/cder/cancer/druglistframe.htm, and The Merck Manual,Seventeenth Ed. 1999, the entire contents of which are herebyincorporated by reference.

Compositions for Administration into a Subject

The ATR kinase inhibitors or pharmaceutical salts thereof may beformulated into pharmaceutical compositions for administration toanimals or humans. These pharmaceutical compositions, which comprise anamount of the ATR inhibitor effective to treat or prevent the diseasesor conditions described herein and a pharmaceutically acceptablecarrier, are another embodiment of the present invention.

The exact amount of compound required for treatment will vary fromsubject to subject, depending on the species, age, and general conditionof the subject, the severity of the infection, the particular agent, itsmode of administration, and the like. The compounds of the invention arepreferably formulated in dosage unit form for ease of administration anduniformity of dosage. The expression “dosage unit form” as used hereinrefers to a physically discrete unit of agent appropriate for thepatient to be treated. It will be understood, however, that the totaldaily usage of the compounds and compositions of the present inventionwill be decided by the attending physician within the scope of soundmedical judgment. The specific effective dose level for any particularpatient or organism will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific compound employed; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts. The term “patient”, as usedherein, means an animal, preferably a mammal, and most preferably ahuman.

In some embodiments, these compositions optionally further comprise oneor more additional therapeutic agents. For example, chemotherapeuticagents or other anti-proliferative agents may be combined with thecompounds of this invention to treat proliferative diseases and cancer.Examples of known agents with which these compositions can be combinedare listed above under the “Combination Therapies” section and alsothroughout the specification. Some embodiments provide a simultaneous,separate or sequential use of a combined preparation.

Modes of Administration and Dosage Forms

The pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), bucally, as an oral or nasal spray, orthe like, depending on the severity of the infection being treated. Incertain embodiments, the compounds of the invention may be administeredorally or parenterally at dosage levels of about 0.01 mg/kg to about 50mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subjectbody weight per day, one or more times a day, to obtain the desiredtherapeutic effect.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar—agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in microencapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, eardrops, and eye drops are also contemplated asbeing within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms can be made by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

The compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes, but is not limited to, subcutaneous,intravenous, intramuscular, intra-articular, intra-synovial,intrasternal, intrathecal, intrahepatic, intralesional and intracranialinjection or infusion techniques. Preferably, the compositions areadministered orally, intraperitoneally or intravenously.

Sterile injectable forms of the compositions of this invention may beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or di-glycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents which are commonlyused in the formulation of pharmaceutically acceptable dosage formsincluding emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers commonly used include, but arenot limited to, lactose and corn starch. Lubricating agents, such asmagnesium stearate, are also typically added. For oral administration ina capsule form, useful diluents include lactose and dried cornstarch.When aqueous suspensions are required for oral use, the activeingredient is combined with emulsifying and suspending agents. Ifdesired, certain sweetening, flavoring or coloring agents may also beadded.

Alternatively, the pharmaceutical compositions of this invention may beadministered in the form of suppositories for rectal administration.These can be prepared by mixing the agent with a suitable non-irritatingexcipient that is solid at room temperature but liquid at rectaltemperature and therefore will melt in the rectum to release the drug.Such materials include, but are not limited to, cocoa butter, beeswaxand polyethylene glycols.

The pharmaceutical compositions of this invention may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may beformulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutical compositions can be formulatedin a suitable lotion or cream containing the active components suspendedor dissolved in one or more pharmaceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions may be formulated asmicronized suspensions in isotonic, pH adjusted sterile saline, or,preferably, as solutions in isotonic, pH adjusted sterile saline, eitherwith or without a preservative such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutical compositions maybe formulated in an ointment such as petrolatum.

The pharmaceutical compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

The amount of protein kinase inhibitor that may be combined with thecarrier materials to produce a single dosage form will vary dependingupon the host treated, the particular mode of administration.Preferably, the compositions should be formulated so that a dosage ofbetween 0.01-100 mg/kg body weight/day of the inhibitor can beadministered to a patient receiving these compositions. More preferably,the compositions should be formulated so that a dosage of between0.01-100 mg/kg body weight/dose of the inhibitor can be administered toa patient receiving these compositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of inhibitor will also depend upon the particular compound in thecomposition.

Administering with another Agent

Depending upon the particular protein kinase-mediated conditions to betreated or prevented, additional drugs, which are normally administeredto treat or prevent that condition, may be administered together withthe compounds of this invention.

Those additional agents may be administered separately, as part of amultiple dosage regimen, from the protein kinase inhibitor-containingcompound or composition. Alternatively, those agents may be part of asingle dosage form, mixed together with the protein kinase inhibitor ina single composition.

Another aspect of this invention is directed towards a method oftreating cancer in a subject in need thereof, comprising the sequentialor co-administration of a compound of this invention or apharmaceutically acceptable salt thereof, and an anti-cancer agent. Insome embodiments, said anti-cancer agent is selected from Platinatingagents, such as Cisplatin, Oxaliplatin, Carboplatin, Nedaplatin, orSatraplatin and other derivatives; Topo I inhibitors, such asCamptothecin, Topotecan, irinotecan/SN38, rubitecan and otherderivatives; Antimetabolites, such as Folic family (Methotrexate,Pemetrexed and relatives); Purine family (Thioguanine, Fludarabine,Cladribine, 6-Mercaptopurine and relatives); Pyrimidine family(Cytarabine, Gemcitabine, 5-Fluorouracil and relatives); Alkylatingagents, such as Nitrogen mustards (Cyclophosphamide, Melphalan,Chlorambucil, mechlorethamine, Ifosfamide, and relatives); nitrosoureas(e.g. Carmustine); Triazenes (Dacarbazine, temozolomide); Alkylsulphonates (e.g. Busulfan); Procarbazine and Aziridines; Antibiotics,such as Hydroxyurea; Anthracyclines (doxorubicin, daunorubicin,epirubicin and other derivatives); Anthracenediones (Mitoxantrone andrelatives); Streptomyces family (Bleomycin, Mitomycin C, actinomycin)and Ultraviolet light.

Another embodiment provides administering a compound of this inventionwith an additional therapeutic agent that inhibits or modulates a baseexcision repair protein. In some embodiments, the base excision repairprotein is selected from UNG, SMUG1, MBD4, TDG, OGG1, MYH, NTH1, MPG,NEIL1, NEIL2, NEIL3 (DNA glycosylases); APE1, APEX2 (AP endonucleases);LIG1, LIG3 (DNA ligases I and III); XRCC1 (LIG3 accessory); PNK, PNKP(polynucleotide kinase and phosphatase); PARP1, PARP2 (Poly(ADP-Ribose)Polymerases); PolB, PolG (polymerases); FEN1 (endonuclease) orAprataxin. In other embodiments, the base excision repair protein isselected from PARP1, PARP2, or Po1B. In yet other embodiments, the baseexcision repair protein is selected from PARP1 or PARP2. In someembodiments, the agent is selected from Olaparib (also known as AZD2281or KU-0059436), Iniparib (also known as BSI-201 or SAR240550), Veliparib(also known as ABT-888), Rucaparib (also known as PF-01367338),CEP-9722, INO-1001, MK-4827, E7016, BMN673, or AZD2461.

Biological Samples

As inhibitors of ATR kinase, the compounds and compositions of thisinvention are also useful in biological samples. One aspect of theinvention relates to inhibiting ATR kinase activity in a biologicalsample, which method comprises contacting said biological sample with acompound described herein or a composition comprising said compound. Theterm “biological sample”, as used herein, means an in vitro or an exvivo sample, including, without limitation, cell cultures or extractsthereof; biopsied material obtained from a mammal or extracts thereof;and blood, saliva, urine, feces, semen, tears, or other body fluids orextracts thereof The term “compounds described herein” includescompounds of formula I.

Inhibition of ATR kinase activity in a biological sample is useful for avariety of purposes that are known to one of skill in the art. Examplesof such purposes include, but are not limited to, blood transfusion,organ-transplantation, and biological specimen storage.

Study of Protein Kinases

Another aspect of this invention relates to the study of protein kinasesin biological and pathological phenomena; the study of intracellularsignal transduction pathways mediated by such protein kinases; and thecomparative evaluation of new protein kinase inhibitors. Examples ofsuch uses include, but are not limited to, biological assays such asenzyme assays and cell-based assays.

The activity of the compounds as protein kinase inhibitors may beassayed in vitro, in vivo or in a cell line. In vitro assays includeassays that determine inhibition of either the kinase activity or ATPaseactivity of the activated kinase. Alternate in vitro assays quantitatethe ability of the inhibitor to bind to the protein kinase and may bemeasured either by radiolabelling the inhibitor prior to binding,isolating the inhibitor/kinase complex and determining the amount ofradiolabel bound, or by running a competition experiment where newinhibitors are incubated with the kinase bound to known radioligands.Detailed conditions for assaying a compound utilized in this inventionas an inhibitor of ATR is set forth in the Examples below.

Another aspect of the invention provides a method for modulating enzymeactivity by contacting a compound described herein with ATR kinase.

Methods of Treatment

In one aspect, the present invention provides a method for treating orlessening the severity of a disease, condition, or disorder where ATRkinase is implicated in the disease state. In another aspect, thepresent invention provides a method for treating or lessening theseverity of an ATR kinase disease, condition, or disorder whereinhibition of enzymatic activity is implicated in the treatment of thedisease. In another aspect, this invention provides a method fortreating or lessening the severity of a disease, condition, or disorderwith compounds that inhibit enzymatic activity by binding to the ATRkinase. Another aspect provides a method for treating or lessening theseverity of a kinase disease, condition, or disorder by inhibitingenzymatic activity of ATR kinase with an ATR kinase inhibitor.

One aspect of the invention relates to a method of inhibiting ATR kinaseactivity in a patient, which method comprises administering to thepatient a compound described herein, or a composition comprising saidcompound. In some embodiments, said method is used to treat or prevent acondition selected from proliferative and hyperproliferative diseases,such as cancer.

Another aspect of this invention provides a method for treating,preventing, or lessening the severity of proliferative orhyperproliferative diseases comprising administering an effective amountof a compound, or a pharmaceutically acceptable composition comprising acompound, to a subject in need thereof In some embodiments, said subjectis a patient. The term “patient”, as used herein, means an animal,preferably a human.

In some embodiments, said method is used to treat or prevent cancer. Insome embodiments, said method is used to treat or prevent a type ofcancer with solid tumors. In yet another embodiment, said cancer isselected from the following cancers: Oral: buccal cavity, lip, tongue,mouth, pharynx; Cardiac: sarcoma (angiosarcoma, fibrosarcoma,rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma andteratoma; Lung: non-small cell, bronchogenic carcinoma (squamous cell orepidermoid, undifferentiated small cell, undifferentiated large cell,adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma,sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;Gastrointestinal: esophagus (squamous cell carcinoma, larynx,adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel or smallintestines (adenocarcinoma, lymphoma, carcinoid tumors, Karposi'ssarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), largebowel or large intestines (adenocarcinoma, tubular adenoma, villousadenoma, hamartoma, leiomyoma), colon, colon-rectum, colorectal; rectum,Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor[nephroblastoma], lymphoma), bladder and urethra (squamous cellcarcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma, biliary passages;Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myeloma, malignant giant cell tumorchordoma, osteochronfroma (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors; Nervous system: skull (osteoma, hemangioma,granuloma, xanthoma, osteitis deformans), meninges (meningioma,meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma,glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform,oligodendroglioma, schwannoma, retinoblastoma, congenital tumors),spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological:uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumorcervical dysplasia), ovaries (ovarian carcinoma [serouscystadenocarcinoma, mucinous cystadenocarcinoma, unclassifiedcarcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma(embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast; Skin:malignant melanoma, basal cell carcinoma, squamous cell carcinoma,Karposi's sarcoma, keratoacanthoma, moles dysplastic nevi, lipoma,angioma, dermatofibroma, keloids, psoriasis, Thyroid gland: papillarythyroid carcinoma, follicular thyroid carcinoma; medullary thyroidcarcinoma, multiple endocrine neoplasia type 2A, multiple endocrineneoplasia type 2B, familial medullary thyroid cancer, pheochromocytoma,paraganglioma; and Adrenal glands: neuroblastoma.

In some embodiments, the cancer is selected from the cancers describedherein. In some embodiments, said cancer is lung cancer, head and neckcancer, pancreatic cancer, gastric cancer, or brain cancer. In otherembodiments, the cancer is selected from a cancer of the lung or thepancreas.

In yet other embodiments, the cancer is selected from non-small celllung cancer, small cell lung cancer, pancreatic cancer, biliary tractcancer, head and neck cancer, bladder cancer, colorectal cancer,glioblastoma, esophageal cancer, breast cancer, hepatocellularcarcinoma, or ovarian cancer.

In some embodiments, the lung cancer is small cell lung cancer and theadditional therapeutic agents are cisplatin and etoposide. In otherexamples, the lung cancer is non-small cell lung cancer and theadditional therapeutic agents are gemcitabine and cisplatin. In yetother embodiments, the non-small cell lung cancer is squamous non-smallcell lung cancer. In another embodiment, the cancer is breast cancer andthe additional therapeutic agent is cisplatin. In other embodiments, thecancer is triple negative breast cancer.

In certain embodiments, an “effective amount” of the compound orpharmaceutically acceptable composition is that amount effective inorder to treat said disease. The compounds and compositions, accordingto the method of the present invention, may be administered using anyamount and any route of administration effective for treating orlessening the severity of said disease.

One aspect provides a method for inhibiting ATR in a patient comprisingadministering a compound described herein as described herein. Anotherembodiment provides a method of treating cancer comprising administeringto a patient a compound described herein, wherein the variables are asdefined herein.

Some embodiments comprising administering to said patient an additionaltherapeutic agent selected from a DNA-damaging agent; wherein saidadditional therapeutic agent is appropriate for the disease beingtreated; and said additional therapeutic agent is administered togetherwith said compound as a single dosage form or separately from saidcompound as part of a multiple dosage form.

In some embodiments, said DNA-damaging agent is selected from ionizingradiation, radiomimetic neocarzinostatin, a platinating agent, a Topo Iinhibitor, a Topo II inhibitor, an antimetabolite, an alkylating agent,an alkyl sulphonates, an antimetabolite, or an antibiotic. In otherembodiments, said DNA-damaging agent is selected from ionizingradiation, a platinating agent, a Topo I inhibitor, a Topo II inhibitor,or an antibiotic.

Examples of Platinating agents include Cisplatin, Oxaliplatin,Carboplatin, Nedaplatin, Satraplatin and other derivatives. Otherplatinating agents include Lobaplatin, and Triplatin. Other platinatingagents include Tetranitrate, Picoplatin, Satraplatin, ProLindac andAroplatin.

Examples of Topo I inhibitor include Camptothecin, Topotecan,irinotecan/SN38, rubitecan and other derivatives. Other Topo Iinhibitors include Belotecan.

Examples of Topo II inhibitors include Etoposide, Daunorubicin,Doxorubicin, Aclarubicin, Epirubicin, Idarubicin, Amrubicin,Pirarubicin, Valrubicin, Zorubicin and Teniposide.

Examples of Antimetabolites include members of the Folic family, Purinefamily (purine antagonists), or Pyrimidine family (pyrimidineantagonists). Examples of the Folic family include methotrexate,pemetrexed and relatives; examples of the Purine family includeThioguanine, Fludarabine, Cladribine, 6-Mercaptopurine, and relatives;examples of the Pyrimidine family include Cytarabine, gemcitabine,5-Fluorouracil (5FU) and relatives.

Some other specific examples of antimetabolites include Aminopterin,Methotrexate, Pemetrexed, Raltitrexed, Pentostatin, Cladribine,Clofarabine, Fludarabine, Thioguanine, Mercaptopurine, Fluorouracil,Capecitabine, Tegafur, Carmofur, Floxuridine, Cytarabine, Gemcitabine,Azacitidine and Hydroxyurea.

Examples of alkylating agents include Nitrogen mustards, Triazenes,alkyl sulphonates, Procarbazine and Aziridines. Examples of Nitrogenmustards include Cyclophosphamide, Melphalan, Chlorambucil andrelatives; examples of nitrosoureas include Carmustine; examples oftriazenes include Dacarbazine and temozolomide; examples of alkylsulphonates include Busulfan.

Other specific examples of alkylating agents include Mechlorethamine,Cyclophosphamide, Ifosfamide, Trofosfamide, Chlorambucil, Melphalan,Prednimustine, Bendamustine, Uramustine, Estramustine, Carmustine,Lomustine, Semustine, Fotemustine, Nimustine, Ranimustine, Streptozocin,Busulfan, Mannosulfan, Treosulfan, Carboquone, ThioTEPA, Triaziquone,Triethylenemelamine, Procarbazine, Dacarbazine, Temozolomide,Altretamine, Mitobronitol, Actinomycin, Bleomycin, Mitomycin andPlicamycin.

Examples of antibiotics include Mitomycin, Hydroxyurea; Anthracyclines,Anthracenediones, Streptomyces family. Examples of Anthracyclinesinclude doxorubicin, daunorubicin, epirubicin and other derivatives;examples of Anthracenediones include Mitoxantrone and relatives;examples of Streptomyces family inclue Bleomycin, Mitomycin C, andactinomycin.

In certain embodiments, said platinating agent is Cisplatin orOxaliplatin; said Topo I inhibitor is Camptothecin; said Topo IIinhibitor is Etoposide; and said antibiotic is Mitomycin. In otherembodiments, said platinating agent is selected from Cisplatin,Oxaliplatin, Carboplatin, Nedaplatin, or Satraplatin; said Topo Iinhibitor is selected from Camptothecin, Topotecan, irinotecan/SN38,rubitecan; said Topo II inhibitor is selected from Etoposide; saidantimetabolite is selected from a member of the Folic Family, the PurineFamily, or the Pyrimidine Family; said alkylating agent is selected fromnitrogen mustards, nitrosoureas, triazenes, alkyl sulfonates,Procarbazine, or aziridines; and said antibiotic is selected fromHydroxyurea, Anthracyclines, Anthracenediones, or Streptomyces family.

In some embodiments, the additional therapeutic agent is ionizingradiation. In other embodiments, the additional therapeutic agent isCisplatin or Carboplatin. In yet other embodiments, the additionaltherapeutic agent is Etoposide. In yet other embodiments, the additionaltherapeutic agent is Temozolomide.

In certain embodiments, the additional therapeutic agent is selectedfrom one or more of the following: Cisplatin, Carboplatin, gemcitabine,Etoposide, Temozolomide, or ionizing radiation.

Another embodiment provides methods for treating pancreatic cancer byadministering a compound described herein in combination with anotherknown pancreatic cancer treatment. One aspect of the invention includesadministering a compound described herein in combination withgemcitabine. In some embodiments, the pancreatic cancer comprises one ofthe following cell lines: PSN-1, MiaPaCa-2 or Panc-1. According toanother aspect, the cancer comprises one of the following primary tumorlines: Panc-M or MRCS.

Another aspect of the invention includes administering a compounddescribed herein in combination with radiation therapy. Yet anotheraspect provides a method of abolishing radiation-induced G2/M checkpointby administering a compound described herein in combination withradiation treatment.

Another aspect provides a method of treating pancreatic cancer byadministering to pancreatic cancer cells a compound described herein incombination with one or more cancer therapies. In some embodiments, thecompound is combined with chemoradiation, chemotherapy, and/or radiationtherapy. As would be understood by one of skill in the art,chemoradiation refers to a treatment regime that includes bothchemotherapy (such as gemcitabine) and radiation. In some embodiments,the chemotherapy is gemcitabine.

Yet another aspect provides a method of increasing the sensitivity ofpancreatic cancer cells to a cancer therapy selected from gemcitabine orradiation therapy by administering a compound described herein incombination with the cancer therapy.

In some embodiments, the cancer therapy is gemcitabine. In otherembodiments, the cancer therapy is radiation therapy. In yet anotherembodiment the cancer therapy is chemoradiation.

Another aspect provides a method of inhibiting phosphorylation of Chk1(Ser 345) in a pancreatic cancer cell comprising administering acompound described herein after treatment with gemcitabine (100 nM)and/or radiation (6 Gy) to a pancreatic cancer cell.

Another aspect provides method of radiosensitizing hypoxic PSN-1,MiaPaCa-2 or PancM tumor cells by administering a compound describedherein to the tumor cell in combination with radiation therapy.

Yet another aspect provides a method of sensitizing hypoxic PSN-1,MiaPaCa-2 or PancM tumor cells by administering a compound describedherein to the tumor cell in combination with gemcitabine.

Another aspect provides a method of sensitizing PSN-1 and MiaPaCa-2tumor cells to chemoradiation by administering a compound describedherein to the tumor cells in combination with chemoradiation.

Another aspect provides a method of disrupting damage-induced cell cyclecheckpoints by administering a compound described herein in combinationwith radiation therapy to a pancreatic cancer cell.

Another aspect provides a method of inhibiting repair of DNA damage byhomologous recombination in a pancreatic cancer cell by administering acompound described herein in combination with one or more of thefollowing treatments: chemoradiation, chemotherapy, and radiationtherapy.

In some embodiments, the chemotherapy is gemcitabine.

Another aspect provides a method of inhibiting repair of DNA damage byhomologous recombination in a pancreatic cancer cell by administering acompound described herein in combination with gemcitabine and radiationtherapy.

In some embodiments, the pancreatic cancer cells are derived from apancreatic cell line selected from PSN-1, MiaPaCa-2 or Panc-1.

In other embodiments, the pancreatic cancer cells are in a cancerpatient.

Another aspect of the invention provides a method of treating non-smallcell lung cancer comprising administering to a patient a compounddescribed herein in combination with one or more of the followingadditional therapeutic agents: Cisplatin or Carboplatin, Etoposide, andionizing radiation. Some embodiments comprise administering to a patienta compound described herein in combination with Cisplatin orCarboplatin, Etoposide, and ionizing radiation. In some embodiments thecombination is Cisplatin, Etoposide, and ionizing radiation. In otherembodiments the combination is Carboplatin, Etoposide, and ionizingradiation.

Another embodiment provides a method of promoting cell death in cancercells comprising administering to a patient a compound described herein,or a composition comprising said compound.

Yet another embodiment provides a method of preventing cell repair ofDNA damage in cancer cells comprising administering to a patient acompound described herein, or a composition comprising said compound.Yet another embodiment provides a method of preventing cell repaircaused by of DNA damage in cancer cells comprising administering to apatient a compound of formula I, or composition comprising saidcompound.

Another embodiment provides a method of sensitizing cells to DNAdamaging agents comprising administering to a patient a compounddescribed herein, or a composition comprising said compound.

In some embodiments, the method is used on a cancer cell having defectsin the ATM signaling cascade. In some embodiments, said defect isaltered expression or activity of one or more of the following: ATM,p53, CHK2, MRE11, RAD50, NBS1, 53BP1, MDC1, H2AX, MCPH1/BRIT1, CTIP, orSMC1. In other embodiments, said defect is altered expression oractivity of one or more of the following: ATM, p53, CHK2, MRE11, RAD50,NBS1, 53BP1, MDC1 or H2AX. According to another embodiment, the methodis used on a cancer, cancer cell, or cell expressing DNA damagingoncogenes.

In another embodiment, the cell is a cancer cell expressing DNA damagingoncogenes. In some embodiments, said cancer cell has altered expressionor activity of one or more of the following: K-Ras, N-Ras, H-Ras, Raf,Myc, Mos, E2F, Cdc25A, CDC4, CDK2, Cyclin E, Cyclin A and Rb.

According to another embodiment, the method is used on a cancer, cancercell, or cell has a defect in a protein involved in base excision repair(“base excision repair protein”). There are many methods known in theart for determining whether a tumor has a defect in base excisionrepair. For example, sequencing of either the genomic DNA or mRNAproducts of each base excision repair gene (e.g., UNG, PARP1, or LIG1)can be performed on a sample of the tumor to establish whether mutationsexpected to modulate the function or expression of the gene product arepresent (Wang et al., Cancer Research 52:4824 (1992)). In addition tothe mutational inactivation, tumor cells can modulate a DNA repair geneby hypermethylating its promoter region, leading to reduced geneexpression. This is most commonly assessed using methylation-specificpolymerase chain reaction (PCR) to quantify methylation levels on thepromoters of base excision repair genes of interest. Analysis of baseexcision repair gene promoter methylation is available commercially(http://www.sabiosciences.com/dna_methylation_product/HTML/MEAH-421A.html).

Finally, the expression levels of base excision repair genes can beassessed by directly quantifying levels of the mRNA and protein productsof each gene using standard techniques such as quantitative reversetranscriptase-coupled polymerase chain reaction (RT-PCR) andimmunhohistochemistry (IHC), respectively (Shinmura et al.,Carcinogenesis 25: 2311 (2004); Shinmura et al., Journal of Pathology225:414 (2011)).

In some embodiments, the base excision repair protein is UNG, SMUG1,MBD4, TDG, OGG1, MYH, NTH1, MPG, NEIL1, NEIL2, NEIL3 (DNA glycosylases);APE1, APEX2 (AP endonucleases); LIG1, LIG3 (DNA ligases I and III);XRCC1 (LIG3 accessory); PNK, PNKP (polynucleotide kinase andphosphatase); PARP1, PARP2 (Poly(ADP-Ribose) Polymerases); PolB, PolG(polymerases); FEN1 (endonuclease) or Aprataxin.

In sorme embodiments, the base excision repair protein is PARP1, PARP2,or PolB. In other embodiments, the base excision repair protein is PARP1or PARP2.

The methods described above (gene sequence, promoter methylation andmRNA expression) may also be used to characterize the status (e.g.,expression or mutation) of other genes or proteins of interesting, suchDNA-damaging oncogenes expressed by a tumor or defects in the ATMsignaling cascade of a cell.

Yet another embodiment provides use of a compound described herein as aradio-sensitizer or a chemo-sensitizer.

Yet other embodiment provides use of a compound of formula I as a singleagent (monotherapy) for treating cancer. In some embodiments, thecompounds of formula I are used for treating patients having cancer witha DNA-damage response (DDR) defect. In other embodiments, said defect isa mutation or loss of ATM, p53, CHK2, MRE11, RAD50, NBS1, 53BP1, MDC1,or H2AX.

Compounds and Compositions for Use

One embodiment provides a compound or composition as described hereinfor use as a radio-sensitizer or a chemo-sensitizer. Another embodimentprovides a compound or composition as described herein for use as asingle agent (monotherapy) for treating cancer.

Another embodiment provides a compound or composition as describedherein for treating patients having cancer with a DNA-damage response(DDR) defect. In some embodiments, said defect is a mutation or loss ofATM, p53, CHK2, MRE11, RAD50, NBS1, 53BP1, MDC1, or H2AX. In otherembodiments, said defect is a mutation or loss of ATM, p53, CHK2, MRE11,RAD50, NBS1, 53BP1, MDC1, H2AX, MCPH1/BRIT1, CTIP, or SMC1.

Another embodiment provides compounds or compositions described hereinfor treating cancer. In some embodiments, the compound or composition isfurther combined with an additional therapeutic agent described herein.In some embodiments, the compound or composition is further combinedwith a DNA damaging agent described herein.

In some embodiments, the cancer has a defect in a pathway describedherein.

Manufacture of Medicaments

One embodiment provides the use of a compound or composition describedherein for the manufacture of a medicament for use as a radio-sensitizeror a chemo-sensitizer. Another embodiment provides the use of a compoundor composition described herein for the manufacture of a medicament forthe manufacture of a medicament for use as a single agent (monotherapy)for treating cancer.

Yet another embodiment provides the use of a compound or compositiondescribed herein for the manufacture of a medicament for the manufactureof a medicament for treating patients having cancer with a DNA-damageresponse (DDR) defect.

In some embodiments, said defect is a mutation or loss of ATM, p53,CHK2, MRE11, RAD50, NBS1, 53BP1, MDC1, or H2AX. In other embodiments,said defect is a mutation or loss of ATM, p53, CHK2, MRE11, RAD50, NBS1,53BP1, MDC1, H2AX, MCPH1/BRIT1, CTIP, or SMC1.

Another embodiment provides the use of a compound or compositiondescribed herein for the manufacture of a medicament for treatingcancer. In some embodiments, the compound or composition is combinedwith an additional therapeutic agent, such as a DNA damaging agent,described herein. In another embodiment, the cancer has a defect in apathway described herein.

EXPERIMENTAL MATERIALS AND METHODS

All commercially available solvents and reagents were used as received.¹H-NMR spectra were recorded at 400 MHz using a Bruker DPX 400instrument. Mass spec. samples were analyzed on a MicroMass QuattroMicro mass spectrometer operated in single MS mode with electrosprayionization. HPLC purity was measured on a Waters Acquity UPLC systemwith a Waters SQD MS instrument equipped with a Waters UPLC BEH C8 1.7μm, 2.1×50 mm column and a Vanguard BEH C8 1.7 μm, 2.1×5 mm guardcolumn.

As used herein, the term “Rt(min)” refers to the HPLC retention time, inminutes, associated with the compound. Unless otherwise indicated, theHPLC methods utilized to obtain the reported retention times are asdescribed below:

HPLC Method

-   Instrument: Waters Acquity UPLC-MS;-   Column: Waters UPLC BEH C8 1.7 μm, 2.1×50 mm with Vanguard BEH C8    1.7 μm, 2.1×5 mm guard column;-   Column temperature: 45° C.;-   Mobile Phase A: 10 mM ammonium formate in water:acetonitrile 95:5,    pH 9;-   Mobile Phase B: acetonitrile;-   Detection: 210-400 nm;-   Gradient: 0-0.40 min: 2% B, 0.40-4.85 min: 2% B to 98% B, 4.85-4.90    min: 98% B to 2% B, 4.90-5.00 min: hold at 2% B;-   Flow rate: 0.6 mL/minute.

Examples and Schemes

The compounds of the disclosure may be prepared in light of thespecification using steps generally known to those of ordinary skill inthe art. Those compounds may be analyzed by known methods, including butnot limited to LCMS (liquid chromatography mass spectrometry) and NMR(nuclear magnetic resonance), described above. The following genericschemes and examples illustrate how to prepare the compounds of thepresent disclosure. The examples are for the purpose of illustrationonly and are not to be construed as limiting the scope of the inventionin any way.

Example 15-(4-isopropylsulfonylphenyl)-3-[3-[4-(2-piperidylmethylamino)phenyl]isoxazol-5-yl]pyrazin-2-amineI-1

Compound I-1 was prepared using Method A, Steps 1-6

Method A:

Step 1: 5-Bromo-3-((trimethylsilyl)ethynyl)pyrazin-2-amine 2

(Trimethylsilyl)acetylene (1.845 g, 2.655 mL, 18.78 mmol) was addeddropwise to a solution of 3,5-dibromopyrazin-2-amine 1 (5 g, 19.77 mmol)in DMF (25 mL) Triethylamine (10.00 g, 13.77 mL, 98.85 mmol), copper(I)iodide (451.7 mg, 2.372 mmol) and Pd(PPh₃)₄ (1.142 g, 0.9885 mmol) werethen added and the resulting solution stirred at RT for 30 minutes. Thereaction mixture was diluted with EtOAc and water and the layersseparated. The aqueous layer was extracted further with EtOAc and thecombined organic layers washed with water, dried (MgSO₄) andconcentrated in vacuo. The residue was purified by column chromatographyeluting with 15% EtOAc/Petroleum ether to give the product as a yellowsolid (3.99 g, 75% Yield). 1H NMR (400.0 MHz, DMSO) δ 0.30 (9H, s), 8.06(IH, s); MS (ES⁺) 271.82.

Step 2: tert-ButylN-tert-butoxycarbonyI-N-[5-bromo-3-((trimethylsilyl)ethyynyl)pyrazin-2-yl]carbamate3

5-Bromo-3-(2-trimethylsilylethynyl)pyrazin-2-amine 2 (2.85 g, 10.55mmol) was dissolved in DCM (89.06 mL) and treated with BOC anhydride(6.908 g, 7.272 mL, 31.65 mmol) followed by DMAP (128.9 mg, 1.055 mmol).The reaction was allowed to stir at ambient temperature for 2 hours. Themixture was then diluted with CH₂Cl₂ and NaHCO₃ and the layersseparated. The aqueous layer was extracted further with CH₂Cl₂, dried(MgSO₄), filtered and concentrated in vacuo. The resultant residue waspurified by column chromatography eluting with dichloromethane to givethe desired product as a colourless oil (4.95 g, 99% Yield). 1H NMR(400.0 MHz, DMSO) δ 0.27 (9H, s), 1.42 (18H, s), 8.50 (1H, s); MS (ES⁺)472.09.

Step 3: tert-ButylN-(3-ethynyl-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)N-tertbutoxycarbonyl-carbamatetert-butyl 4

N-[5-Bromo-3-(2-trimethylsilylethynyl)pyrazin-2-yl]-N-tertbutoxycarbonylcarbamate 3 (3 g, 6.377 mmol) and(4-isopropylsulfonylphenyl)boronic acid (1.491 g, 6.536 mmol) weredissolved in MeCN/water (60/12 mL). K₃PO₄ (2.706 g, 12.75 mmol) wasadded and the reaction mixture was degassed with a flow of nitrogen (5cycles). Pd[P(tBu)₃]₂ (162.9 mg, 0.3188 mmol) was added and theresulting mixture was stirred at room temperature for 1 h. The reactionmixture was poured quickly into a mixture of ethyl acetate (500 mL),water (90 mL) and 1% aqueous sodium metabisulphite at 4° C., shaken welland the layer separated. The organic fraction was dried over MgSO₄,filtered and the filtrate was treated with 3-mercaptopropyl ethylsulphide on silica (0 8 mmol/g, 1 g), pre-absorbed onto silica gel thenpurified by column chromatography on silica gel eluting with 30-40%EtOAc/petroleum ether. The solvents were concentrated in vacuo to leavethe product as a yellow viscous oil that was triturated with petroleumether to yield the product as beige crystals (1.95 g, 61% Yield); 1H NMR(400 MHz, DMSO) δ 1.20 (m, 6H), 1.39 (s, 18H), 3.50 (m, 1H), 5.01 (s,1H), 8.03 (m, 2H), 8.46 (m, 2H) and 9.37 (s, IH).

Step 4: tert-butylN-tert-butoxycarbonyl-N-[5-(4-isopropylsulfonylphenyl)-3-[3-(4-nitrophenyl)isoxazol-5-yl]pyrazin-2-yl]carbamate11

Triethylamine (302.7 mg, 416.9 μL, 2.991 mmol) was added dropwise to asolution containing tert-butylN-tert-butoxycarbonyl-N-[3-ethynyl-5-(4-isopropylsulfonylphenyl)pyrazin-2-yl]carbamate(1 g, 1.994 mmol) and (1E)-N-hydroxy-4-nitro-benzimidoyl chloride (599.9mg, 2.991 mmol) in THF (10 mL) and the resulting mixture stirred at rtfor 30 min and then heated at 65° C. for 4 h. The reaction mixture wascooled to rt and then was concentrated in vacuo. The residue waspurified by column chromatography (ISCO Companion, 24 g column) elutingwith 0-65% EtOAc/ petroleum ether, product fractions were combined andconcentrated in vacuo to leave the desired product as a yellow solid(552 mg, 42% yield); 1H NMR (400 MHz, DMSO) δ 0.22 (m, 6H), 0.35 (s,18H), 2.59 (m, 1H), 7.11 (d, J=8.0 Hz, 2H), 7.28 (s, 1H), 7.38 (d, J=4.0Hz, 2H), 7.46 (d, J=8.0 Hz, 2H), 7.67 (d, J=8.0 Hz, 2H) and 8.57 (s, 1H)ppm; MS (ES⁺) 510.2 (M-BOC-tBu).

Step 5: tert-butylN-[3-[3-(4-aminophenyl)isoxazol-5-yl]-5-(4-isopropylsulfonylphenyl)pyrazin-2-yl]-N-tert-butoxycarbonyl-carbamate12

Tin (II) chloride (499.4 mg, 2.213 mmol) was added to a stirred solutionof tert-butylN-tert-butoxycarbonyl-N-[5-(4-isopropylsulfonylphenyl)-3-[3-(4-nitrophenyl)isoxazol-5-yl]pyrazin-2-yl]carbamate(491 mg, 0.7376 mmol) in ethanol (15 mL) and the resulting solutionheated at 60° C. for 16 h. The reaction mixture was cooled rt andconcentrated in vacuo. The residue was partitioned between ethyl acetate(10 mL) and 1M NaOH_((aq)) (5 mL) and the residue that formed wasfiltered off. The layers were then separated and the aqueous layer wasextracted further with ethyl acetate (2×10 mL) and the combined organicextracts were washed with brine (1×10 mL), dried over MgSO₄ andconcentrated in vacuo. The material was purified by columnchromatography on silica (ISCO Companion, 12 g column) eluting with0-100% EtOAc with petroleum ether. The product fractions were combinedand concentrated in vacuo to leave the title compound as a yellow/orangesolid (286 mg, 61% yield); 1H NMR (400 MHz, CDCl₃) δ 1.37 (m, 6H), 1.42(s, 18H), 3.28 (m, 1H), 3.95 (br s, 2H), 6.79 (m, 2H), 7.30 (s, 1H),7.72 (d, J=8.0 Hz, 1H), 8.10 (m, 2H), 8.36 (m, 2H) and 9.06 (s, 1H) ppm;MS (ES⁺) 636.3 (M+H)

Step 6:5-(4-isopropylsulfonylphenyl)-3-[3-[4-(2-piperidylmethylamino)phenyl]isoxazol-5-yl]pyrazin-2-amineI-1

To a solution of tert-butyl 2-formylpiperidine-l-carboxylate (18.45 mg,0.08652 mmol) and tert-butylN-[3-[3-(4-aminophenyl)isoxazol-5-yl]-5-(4-isopropylsulfonylphenyl)pyrazin-2-yl]-N-tert-butoxycarbonyl-carbamate(50 mg, 0.07865 mmol) in CH₂Cl₂ (5.0 mL) at 0° C. was added acetic acid(5.196 mg, 4.920 μL, 0.08652 mmol) and sodium triacetoxyborohydride(28.34 mg, 0.1337 mmol) and the resulting solution stirred at 0-10° C.for 2 h. The reaction mixture was diluted with saturated aqueous sodiumhydrogen carbonate solution (5 mL) and dichloromethane (10 mL) and thelayers separated. The aqueous layer was extracted further withdichloromethane (2×5 mL) and the combined organic extracts were thendried over MgSO ₄ and concentrated in vacuo. The residue was taken up inCH₂Cl₂ (3.0 mL) and TFA (44.83 mg, 30.29 μL, 0.3932 mmol) was added andthe resulting solution stirred at rt overnight. The reaction mixture wasconcentrated in vacuo and purified by reverse phase preparative HPLC[Waters Sunfire C18, 100 Å column, 5 M, 19 mm×150 mm, (solvent A: 0.05%TFA in water; solvent B: 0.05% TFA in CH₃CN) over 16 minutes at 20mL/min] The product fractions were collected and lyopholised to leavethe title compound as a yellow solid (30.5 mg, 58% yield); 1H NMR (400MHz, DMSO) δ 1.19 (d, 6H), 1.40-1.59 (m, 3H), 1.79 (m, 2H), 1.96 (m,1H), 2.88 (m, 1H), 3.24 masked signal, 3.47 (m, 1H), 6.37 (t, 1H), 6.79(m, 2H), 7.16 (br s, 2H), 7.64 (s, 1H), 7.78 (m, 2H), 7.94 (m, 2H), 8.28(br s, 1H), 8.38 (m, 2H), 8.52 (br s, 1H) and 8.94 (s, 1H) ppm; MS (ES⁺)533.3 (M+H), (ES⁻) 531.2 (M−H)

Compounds I-2 to I-4, described below, were all prepared using a methodsimilar to the one described for Compound I-1 above.

Example 23-[3-[4-(azetidin-2-ylmethylamino)phenyl]isoxazol-5-yl]-5-(4-isopropylsulfonylphenyl)pyrazin-2-amineI-2

1H NMR (400 MHz, DMSO) d 1.19 (d, 6H), 2.29 (m, 1H), 3.52 (m, 2H), 3.66(m, 1H), 3.82 (m, 1H), 3.94 (m, 1H), 4.54 (m, 1H), 6.28 (m, 1H),6.70-6.81 (m, 2H), 7.16 (br s, 2H), 7.67 (s, 1H), 7.79 (m, 2H), 7.95 (m,2H), 8.37 (m, 2H), 8.59 (br s, 1H) and 8.93 (m, 1H) ppm; MS (ES⁺) 505.3(M+H).

Example 3(S)-5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-((pyrrolidin-2-ylmethyl)amino)phenyl)isoxazol-5-yl)pyrazin-2-amine1-3

1H NMR (400 MHz, DMSO) d 1.19 (d, 6H), 1.67-1.73 (d, 1H), 1.89-2.00 (m,2H), 2.11-2.16 (m, 1H), 3.20 (m, 2H), 3.39 (m, 2H), 3.49 (m, 1H), 3.70(m, 1H), 6.31 (t, 1H), 6.79 (m, 2H), 7.14 (br s, 2H), 7.64 (s, 1H), 7.79(m, 2H), 7.94 (m, 2H), 8.38 (m, 2H) and 8.94 (s, 1H) ppm; MS (ES⁺) 519.1(M+H).

Example 4(R)-5-(4-(isopropylsulfonyl)phenyl)-3-(3-(4-((pyrrolidin-2-ylmethyl)amino)phenyl)isoxazol-5-yl)pyrazin-2-amine1-4

1H NMR (400 MHz, DMSO) d 1.19 (d, 6H), 1.65-1.73 (m, 1H), 1.89-2.00 (m,2H), 2.11-2.18 (m, 1H), 3.16-3.27 (m, 2H), 3.45 (m, 1H), 3.72 (m, 1H),6.32 (br s, 1H), 6.79 (m, 2H), 7.16 (br s, 2H), 7.64 (s, 1H), 7.79 (m,2H), 7.93 (m, 2H), 8.37-8.43 (m, 3H) and 8.93 (m, 2H) ppm; MS (ES⁺)519.3 (M+H).

Compound I-5 was prepared using the method described below.

Example 55-(4-isopropylsulfonylphenyl)-3-[3-[4-(pyrrolidin-2-ylmethylamino)phenyl]isoxazol-5-yl]pyrazin-2-amine1-5

Compound I-5 was prepared using Method B, Steps 1-3

Method B:

Step 1: 4-iodobenzaldehyde oxime 15

To a stirred suspension of 4-iodobenzaldehyde (2000 mg, 8.620 mmol) inethanol (9.000 mL) cooled in an ice bath, hydroxylamine (569.4 μL of 50%w/v, 8.620 mmol) was added and the reaction was removed from ice bathand stirred at rt for 1 h. After this time the reaction mixture washeated at 50° C. for 1 h and then cooled to rt and concentrated in vacuoto leave the title compound as a white solid (2.03 g, 95% yield); 1H NMR(400 MHz, DMSO) d 7.38 (m, 2H), 7.76 (m, 2H), 8.11 (s, 1H) and 11.37 (s,1H) ppm; MS (ES⁺) 248.1.

Step 2:3-(3-(4-iodophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-amine16

To a solution of 4-iodobenzaldehyde oxime (640 mg, 2.591 mmol) in DMF(8.0 mL) was added N-chlorosuccinimde (372 mg, 2.786 mmol) and theresulting mixture was heated at 55° C. for 30 min. The reaction mixturewas cooled to rt and was partitioned between ethyl acetate (10 mL) andwater (10 mL) and the layers separated. The aqueous layer was extractedfurther with ethyl acetate (2×10 mL) and the combined organic extractswere washed sequentially with water (1×10 mL) and brine (1×10 mL), dried(MgSO₄) and concentrated in vacuo to leave theN-hydroxy-4-iodobenzimidoyl chloride as a colourless oil. The oil wasthen dissolved in THF (16.00 mL) and tert-butylN-tert-butoxycarbonyl-N-[3-ethynyl-5-(4-isopropylsulfonylphenyl)pyrazin-2-yl]carbamate(800 mg, 1.595 mmol) and triethylamine (322.8 mg, 444.6 μL, 3.190 mmol)were added sequentially and the resulting mixture was heated at 55° C.overnight. The reaction mixture was cooled to rt and concentrated invacuo and the residue partitioned between CH₂Cl₂ (20 mL) and water (20mL) and the layers separated. The aqueous layer was extracted furtherwith CH₂Cl₂ and the combined organic extracts were dried over MgSO₄ andconcentrated in vacuo to leave an oil which was purified by columnchromatography on silica (ISCO Companion, 12 g column) eluting with10-50% EtOAc with petroleum ether. Product fractions were combined andconcentrated in vacuo to leave the product as a pale yellow oil. The oilwas dissolved in CH₂Cl₂ (16 mL) and TFA (3 mL) was added and theresulting solution stirred at rt for 2 h. The reaction mixture wasconcentrated in vacuo and the title compound 16 used directly in thenext step without further purification (505 mg, 58% yield); 1H NMR (400MHz, DMSO) d 1.18 (d, 6H), 3.46 (m, 1H), 7.82 (m, 3H), 7.95 (m, 4H),8.37 (m, 2H) and 8.95 (s, 1H) ppm; MS (ES⁺) 547.0

Step 3:5-(4-isopropylsulfonylphenyl)-3-[3-[4-(pyrrolidin-2-ylmethylamino)phenyl]isoxazol-5-yl]pyrazin-2-amine1-5

A reaction mixture containing CuI (43.57 mg, 0.2288 mmol), Cs₂CO₃ (149.1mg, 0.4576 mmol), 2-acetylcyclohexanone (64.15 mg, 0.4576 mmol),tert-butyl 2-(aminomethyl)pyrrolidine-1-carboxylate (137.5 mg, 0.6864mmol) and3-(3-(4-iodophenyl)isoxazol-5-yl)-5-(4-(isopropylsulfonyl)phenyl)pyrazin-2-amine(125 mg, 0.2288 mmol) in DMF (7.500 mL) was heated at 90° C. overnight.The reaction mixture was cooled to rt and partitioned between ethylacetate (5 mL) and water (5 mL) and the layers separated. The aqueouslayer was extracted further with ethyl acetate (2×5 mL) and the combinedorganic extracts were washed sequentially with water (1×5 mL) and brine(1×5 mL), dried over MgSO₄ and concentrated in vacuo which yielded anoil. The oil was purified by reverse phase preparative HPLC [WatersSunfire C18, 100 Å column, 5 M, 19 mm×150 mm, (solvent A: 0.05% TFA inwater; solvent B: 0.05% TFA in CH₃CN) over 16 minutes at 20 mL/min]. Theproduct fractions were combined and concentrated to leave a yellowsolid. The solid was dissolved in CH₂Cl₂(10 mL) and TFA (2 mL) was addedand the resulting solution stirred at rt for 5 h. The reaction mixturewas concentrated in vacuo and the residue partitioned between CH₂Cl₂ (5mL) and water (5 mL) and the combined organic extracts were dried overMgSO₄ and concentrated in vacuo which was purified by reverse phasepreparative HPLC [Waters Sunfire C18, 100 Å column, 5 M, 19 mm×150 mm,(solvent A: 0.05% TFA in water; solvent B: 0.05% TFA in CH₃CN) over 16minutes at 20 mL/min]. The product fractions were combined andconcentrated to leave the title compound as a yellow solid (25 mg, 15%yield); 1H NMR (400.0 MHz, DMSO) d 1.19 (d, J=6.9 Hz, 6H), 1.68 (dd,J=8.2, 12.7 Hz, 1H), 1.93 (m, 2H), 2.15-2.11 (m, 1H), 3.23 (m, 2H),3.45-3.39 (m, 1H), 3.56 (m, 1H), 3.71 (m, 2H), 6.80 (d, J=8.7 Hz, 2H),7.18 (s, 1H), 7.65 (s, 1H), 7.80 (d, J=8.7 Hz, 2H), 7.94 (d, J=8.5 Hz,2H), 8.38 (d, J=8.5 Hz, 2H), 8.47 (bs, 1H) and 8.94 (s, 2H) ppm; MS(ES⁺) 519.

The following compounds were all prepared using a method similar to theone described for Compound I-5 above.

Example 6N-(4-(5-(3-amino-6-(4-(isopropylsulfonyl)phenyl)pyrazin-2-yl)isoxazol-3-yl)phenyl)-N,N-dimethylethane-1,2-diamineI-6

1H NMR (400.0 MHz, DMSO) d 1.19 (d, J=6.8 Hz, 6H), 2.86 (d, J=2.1 Hz,6H), 3.28 (m, 2H), 3.51-3.44 (m, 3H), 6.79 (d, J=8.7 Hz, 2H), 7.18 (s,2H), 7.65 (s, 1H), 7.80 (d, J=8.7 Hz, 2H), 7.94 (d, J=8.6 Hz, 2H), 8.38(d, J=8.6 Hz, 2H), 8.94 (s, 1H) and 9.05 (bs, 1H) ppm; MS (ES⁺) 507.0

Example 7 Cellular ATR Inhibition Assay

Compounds can be screened for their ability to inhibit intracellular ATRusing an immunofluorescence microscopy assay to detect phosphorylationof the ATR substrate histone H2AX in hydroxyurea treated cells. HT29cells are plated at 14,000 cells per well in 96-well black imagingplates (BD 353219) in McCoy's 5A media (Sigma M8403) supplemented with10% foetal bovine serum (JRH Biosciences 12003), Penicillin/Streptomycinsolution diluted 1:100 (Sigma P7539), and 2 mM L-glumtamine (SigmaG7513), and allowed to adhere overnight at 37° C. in 5% CO₂. Compoundsare then added to the cell media from a final concentration of 25 μM in3-fold serial dilutions and the cells are incubated at 37° C. in 5% CO₂.After 15 min, hydroxyurea (Sigma H8627) is added to a finalconcentration of 2 mM.

After 45 min of treatment with hydroxyurea, the cells are washed in PBS,fixed for 10 min in 4% formaldehyde diluted in PBS (Polysciences Inc18814), washed in 0.2% Tween-20 in PBS (wash buffer), and permeabilisedfor 10min in 0.5% Triton X-100 in PBS, all at room temperature. Thecells are then washed once in wash buffer and blocked for 30 min at roomtemperature in 10% goat serum (Sigma G9023) diluted in wash buffer(block buffer). To detect H2AX phosphorylation levels, the cells arethen incubated for 1 h at room temperature in primary antibody (mousemonoclonal anti-phosphorylated histone H2AX Ser139 antibody; Upstate05-636) diluted 1:250 in block buffer. The cells are then washed fivetimes in wash buffer before incubation for lh at room temperature in thedark in a mixture of secondary antibody (goat anti-mouse Alexa Fluor 488conjugated antibody; Invitrogen A11029) and Hoechst stain (InvitrogenH3570); diluted 1:500 and 1:5000, respectively, in wash buffer. Thecells are then washed five times in wash buffer and finally 100 ul PBSis added to each well before imaging.

Cells are imaged for Alexa Fluor 488 and Hoechst intensity using the BDPathway 855 Bioimager and Attovision software (BD Biosciences, Version1.6/855) to quantify phosphorylated H2AX Ser139 and DNA staining,respectively. The percentage of phosphorylated H2AX-positive nuclei in amontage of 9 images at 20× magnification is then calculated for eachwell using BD Image Data Explorer software (BD Biosciences Version2.2.15). Phosphorylated H2AX-positive nuclei are defined asHoechst-positive regions of interest containing Alexa Fluor 488intensity at 1.75-fold the average Alexa Fluor 488 intensity in cellsnot treated with hydroxyurea. The percentage of H2AX positive nuclei isfinally plotted against concentration for each compound and IC50s forintracellular ATR inhibition are determined using Prism software(GraphPad Prism version 3.0cx for Macintosh, GraphPad Software, SanDiego Calif., USA).

The compounds described herein can also be tested according to othermethods known in the art (see Sarkaria et al, “Inhibition of ATM and ATRKinase Activities by the Radiosensitizing Agent, Caffeine: CancerResearch 59: 4375-5382 (1999); Hickson et al, “Identification andCharacterization of a Novel and Specific Inhibitor of theAtaxia-Telangiectasia Mutated Kinase ATM” Cancer Research 64: 9152-9159(2004); Kim et al, “Substrate Specificities and Identification ofPutative Substrates of ATM Kinase Family Members” The Journal ofBiological Chemistry, 274(53): 37538-37543 (1999); and Chiang et al,“Determination of the catalytic activities of mTOR and other members ofthe phosphoinositide-3-kinase-related kinase family” Methods Mol. Biol.281:125-41 (2004)).

Example 8 ATR Inhibition Assay

Compounds were screened for their ability to inhibit ATR kinase using aradioactive-phosphate incorporation assay. Assays were carried out in amixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl₂ and 1 mM DTT. Finalsubstrate concentrations were 10 μM [γ-33P]ATP (3 mCi 33P ATP/mmol ATP,Perkin Elmer) and 800 μM target peptide (ASELPASQPQPFSAKKK (SEQ. ID NO:1)).

Assays were carried out at 25° C. in the presence of 5 nM full-lengthATR. An assay stock buffer solution was prepared containing all of thereagents listed above, with the exception of ATP and the test compoundof interest. 13.5 μL of the stock solution was placed in a 96 well platefollowed by addition of 2 μL of DMSO stock containing serial dilutionsof the test compound (typically starting from a final concentration of15 μM 3-fold serial dilutions) in duplicate (final DMSO concentration7%). The plate was pre-incubated for 10 minutes at 25° C. and thereaction initiated by addition of 15 μL [γ-33P]ATP (final concentration10 μM).

The reaction was stopped after 24 hours by the addition of 30 μL 0.1Mphosphoric acid containing 2 mM ATP. A multiscreen phosphocellulosefilter 96-well plate (Millipore, Cat no. MAPHN0B50) was pretreated with100 μL 0.2M phosphoric acid prior to the addition of 45 μL of thestopped assay mixture. The plate was washed with 5×200 μL 0.2Mphosphoric acid. After drying, 100 μL Optiphase ‘SuperMix’ liquidscintillation cocktail (Perkin Elmer) was added to the well prior toscintillation counting (1450 Microbeta Liquid Scintillation Counter,Wallac).

After removing mean background values for all of the data points,Ki(app) data were calculated from non-linear regression analysis of theinitial rate data using the Prism software package (GraphPad Prismversion 3.0cx for Macintosh, GraphPad Software, San Diego, Calif., USA).

Example 9 Cisplatin Sensitization Assay

Compounds can be screened for their ability to sensitize HCT116colorectal cancer cells to Cisplatin using a 96 h cell viability (MTS)assay. HCT116 cells, which possess a defect in ATM signaling toCisplatin (see, Kim et al.; Oncogene 21:3864 (2002); see also, Takemuraet al.; JBC 281:30814 (2006)) are plated at 470 cells per well in96-well polystyrene plates (Costar 3596) in 150 μl of McCoy's 5A media(Sigma M8403) supplemented with 10% foetal bovine serum (JRH Biosciences12003), Penicillin/Streptomycin solution diluted 1:100 (Sigma P7539),and 2 mM L-glumtamine (Sigma G7513), and allowed to adhere overnight at37° C. in 5% CO₂. Compounds and Cisplatin are then both addedsimultaneously to the cell media in 2-fold serial dilutions from a topfinal concentration of 10 μM as a full matrix of concentrations in afinal cell volume of 200 μl and the cells are then incubated at 37° C.in 5% CO₂. After 96 h, 40 μl of MTS reagent (Promega G358a) is added toeach well and the cells are incubated for lh at 37° C. in 5% CO₂.Finally, absorbance is measured at 490 nm using a SpectraMax Plus 384reader (Molecular Devices) and the concentration of compound required toreduce the IC50 of Cisplatin alone by at least 3-fold (to 1 decimalplace) can be reported.

Example 10 Single Agent HCT116 Activity

Compounds can be screened for single agent activity against HCT116colorectal cancer cells using a 96 h cell viability (MTS) assay. HCT116are plated at 470 cells per well in 96-well polystyrene plates (Costar3596) in 150 μl of McCoy's 5A media (Sigma M8403) supplemented with 10%foetal bovine serum (JRH Biosciences 12003), Penicillin/Streptomycinsolution diluted 1:100 (Sigma P7539), and 2 mM L-glumtamine (SigmaG7513), and allowed to adhere overnight at 37° C. in 5% CO₂. Compoundsare then added to the cell media in 2-fold serial dilutions from a topfinal concentration of 10 μM as a full matrix of concentrations in afinal cell volume of 200 μl, and the cells are then incubated at 37° C.in 5% CO₂. After 96 h, 40 μl of MTS reagent (Promega G358a) is added toeach well and the cells are incubated for lh at 37° C. in 5% CO₂.Finally, absorbance is measured at 490 nm using a SpectraMax Plus 384reader (Molecular Devices) and IC50 values can be calculated.

Compound Analytical Data Cmpd LCMS LCMS No. ES+ (Rt min) HNMR I-1 533.30.85 (DMSO) d 1.19 (6H, d), 1.40-1.59 (3H, m), 1.79 (2H, m), 1.96 (1H,m), 2.88 (1H, m), 3.24 masked signal, 3.47 (1H, m), 6.37 (1H, t), 6.79(2H, m), 7.16 (2H, br s), 7.64 (1H, s), 7.78 (2H, m), 7.94 (2H, m), 8.28(1H, br s), 8.38 (2H, m), 8.52 (1H, br s) and 8.94 (1H, s) ppm I-2 505.30.81 (DMSO) d 1.19 (6H, d), 2.29 (1H, m), 3.52 (2H, m), 3.66 (1H, m),3.82 (1H, m), 3.94 (1H, m), 4.54 (1H, m), 6.28 (1H, m), 6.70-6.81 (2H,m), 7.16 (2H, br s), 7.67 (1H, s), 7.79 2H, m), 7.95 (2H, m), 8.37 (2H,m), 8.59 (1H, br s) and 8.93 (1H, m) ppm I-3 519.1 0.81 (DMSO) d 1.19(6H, d), 1.67-1.73 (1H, d), 1.89-2.00 (2H, m), 2.11-2.16 (1H, m), 3.20(2H, m), 3.39 (2H, m), 3.49 (1H, m), 3.70 (1H, m), 6.31 (1H, t), 6.79(2H, m), 7.14 (2H, br s), 7.64 (1H, s), 7.79 (2H, m), 7.94 (2H, m), 8.38(2H, m) and 8.94 (1H, s) ppm I-4 519.3 0.83 (DMSO) d 1.19 (6H, d),1.65-1.73 (m, 1H), 1.89-2.00 (m, 2H), 2.11-2.18 (m, 1H), 3.16-3.27 (m,2H), 3.45 (m, 1H), 3.72 (m, 1H), 6.32 (br s, 1H), 6.79 (m, 2H), 7.16 (brs, 2H), 7.64 (s, 1H), 7.79 (m, 2H), 7.93 (m, 2H), 8.37-8.43 (m, 3H) and8.93 (m, 2H) ppm I-5 519.0 1.02 HNMR (400.0 MHz, DMSO) d 8.94 (s, 2H),8.47 (bs, 1H), 8.38 (d, J = 8.5 Hz, 2H), 7.94 (d, J = 8.5 Hz, 2H), 7.80(d, J = 8.7 Hz, 2H), 7.65 (s, 1H), 7.18 (s, 1H), 6.80 (d, J = 8.7 Hz,2H), 3.71 (m, 2H), 3.56 (m, 1H), 3.45-3.39 (m, 1H), 3.23 (m, 2H),2.15-2.11 (m, 1H), 1.93 (m, 2H), 1.68 (dd, J = 8.2, 12.7 Hz, 1H) and1.19 (d, J = 6.9 Hz, 6H) ppm I-6 507.0 0.84 HNMR (400.0 MHz, DMSO) d9.05 (bs, 1H), 8.94 (s, 1H), 8.38 (d, J = 8.6 Hz, 2H), 7.94 (d, J = 8.6Hz, 2H), 7.80 (d, J = 8.7 Hz, 2H), 7.65 (s, 1H), 7.18 (s, 2H), 6.79 (d,J = 8.7 Hz, 2H), 3.51-3.44 (m, 3H), 3.28 (m, 2H), 2.86 (d, J = 2.1 Hz,6H) and 1.19 (d, J = 6.8 Hz, 6H) ppm

Compound Biology Single agent ATR ATR Cisplatin Cmpd HT116 inhibitioncellular sensitization No. IC50 (nM) Ki (nM) IC50 (nM) (nM) I-1 2100.635 62 156 I-2 140 0.600 40 78 I-3 83 0.490 14 20 I-4 52 0.350 13 39I-5 26 <0.100 <10 20 I-6 720 0.405 260 78

While we have described a number of embodiments of this invention, it isapparent that our basic examples may be altered to provide otherembodiments that utilize the compounds, methods, and processes of thisinvention. Therefore, it will be appreciated that the scope of thisinvention is to be defined by the appended claims rather than by thespecific embodiments that have been represented by way of exampleherein.

We claim:
 1. A compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein: R¹ isindependently selected from a C₁₋₆ aliphatic, a C₃₋₆ cycloaliphatic, ora 3-6 membered heterocyclyl having 1-2 heteroatoms selected from O, N,or S; R² is independently selected from H, C₁₋₃aliphatic or aC₃₋₆cycloaliphatic; R³ is independently selected from H, C₁₋₆aliphatic,C₃₋₆cycloaliphatic, or a 3-6 membered heterocyclyl having 1-2heteroatoms selected from O, N, or S; R⁴ is independently selected fromH, C₁₋₆aliphatic, C₃₋₆cycloaliphatic, or a 3-6 membered heterocyclylhaving 1-2 heteroatoms selected from O, N, or S; or R² and R³, togetherwith the atoms to which they are attached, form an optionallysubstituted 4-7 membered heterocyclic ring having 1-2 heteroatomsindependently selected from O, N, or S; or R³ and R⁴, together with thenitrogen atom to which they are attached, form an optionally substituted4-7 membered heterocyclic ring independently selected from O, N, or S.2. The compound of claim 1, wherein R¹ is a C₁₋₆aliphatic.
 3. Thecompound according to claim 1, wherein R¹ is an isopropyl.
 4. Thecompound according to claim 3, wherein R² and R³, together with theatoms to which they are attached, form an optionally substituted 4-7membered. heterocyclic ring having 1-2 heteroatoms independentlyselected from O, N, or S.
 5. The compound according to claim 4, whereinR² and R³, together with the atoms to which they are attached, form anoptionally substituted 4-7 membered heterocyclic ring selected frompyrrolidinyl, azetidinyl, or piperidinyl.
 6. The compound according toclaim 1, wherein R² is H.
 7. The compound according to claim 1, whereinR⁴ is H or C₁₋₆aliphatic.
 8. The compound according to claim 7, whereinR⁴ is H.
 9. The compound according to claim 7, wherein R⁴ is methyl. 10.The compound according to claim 1, wherein R³ is C₁₋₆aliphatic.
 11. Thecompound according to claim 9, wherein R³ is methyl.
 12. Apharmaceutical composition comprising a compound according to claim 1and a pharmaceutically acceptable carrier.
 13. A compound selected fromthe following:


14. A method of inhibiting ATR in a biological sample comprising thestep of contacting a compound of claim 1 with said biological sample.15. The method of claim 14, wherein said biological sample is a cell.16. A method of sensitizing cells to cisplatin comprising administeringto a patient a compound of claim 1.